Method of cell culture

ABSTRACT

A method of cell culture comprising providing cells in a cell culture medium to start a cell culture process, and,
     maintaining at least one metabolite selected from 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate, indolelactate, indolecarboxylic acid, homocysteine, 2-hydroxybutyric acid, isovalerate and formate below a concentration C1 in the cell culture medium, wherein C1 is 3 mM and/or   (ii) maintaining at least one amino acid selected from phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine below a concentration C2 in the cell culture medium, wherein C2 is 2 mM.

FIELD OF THE INVENTION

The invention relates to a method of cell culture where theconcentration of one or more of phenylalanine, tyrosine, tryptophan,methionine, leucine, serine, threonine and glycine and/or one or more of3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate,indolelactate, indolecarboxylic acid, homocysteine, 2-hydroxybutyricacid, isovalerate and formate are maintained at low levels in the cellculture medium. The invention also relates to a method of cell culturefor improving cell growth and productivity, in particular in fed-batchculture of mammalian cells at high cell density. The invention alsorelates to media for use in said methods.

BACKGROUND OF THE INVENTION

Proteins have become increasingly important as diagnostic andtherapeutic agents. In most cases, proteins for commercial applicationsare produced in cell culture, from cells that have been engineeredand/or selected to produce unusually high levels of a particular proteinof interest. Optimization of cell culture conditions is important forsuccessful commercial production of proteins. Mammalian cells haveinefficient metabolism which causes them to consume large amounts ofnutrients and convert a significant amount of them to byproducts. Thebyproducts are released into the culture and accumulate over the courseof the culture. Lactate and ammonia, known to be the conventionalinhibitors of cells in culture, are the two major byproducts of cellularmetabolism that accumulate to high levels in culture and beyond certainconcentrations, they start inhibiting the growth and productivity ofcells in culture. Cell culture methods aimed at reducing the amount oflactate and ammonia in the cell culture medium have been developed andcan increase the growth and the productivity of mammalian cells. Thecell growth, however, still slows down even when concentrations oflactate and ammonia are kept low, thereby limiting the maximum celldensity and productivity of the cells.

Therefore, there is a need for the development of improved cell culturesystems for optimum production of proteins. In particular there is aneed for cell culture methods providing an increased viable cell densityand/or titer.

SUMMARY OF THE INVENTION

The invention relates to a method of cell culture comprising

(i) providing cells in a cell culture medium to start a cell cultureprocess, and,(ii) maintaining at least one metabolite selected from3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate,indolelactate, indolecarboxylic acid, homocysteine, 2-hydroxybutyricacid, isovalerate and formate below a concentration C1 in the cellculture medium, wherein C1 is 3 mM and/ormaintaining at least one amino acid selected from phenylalanine,tyrosine, tryptophan, methionine, leucine, serine, threonine and glycinebelow a concentration C2 in the cell culture medium wherein C2 is 2 mM.

The invention also relates to a cell culture medium comprising lowconcentration of one or more of phenylalanine, tyrosine, tryptophan,methionine leucine, serine, threonine and glycine.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows the growth characteristics and metabolic profiles of CHOcells in conventional fed-batch process. Reported data includes viablecell densities (closed squares), culture lactate levels (closedtriangles) and ammonia levels (closed diamonds). Also, reported is theharvest titer (day 12 protein concentration).

FIG. 1B shows the growth characteristics and metabolic profiles of CHOcells in a HiPDOG process. Reported data includes viable cell densities(closed squares), culture lactate levels (closed triangles) and ammonialevels (closed diamonds). Also, reported is the harvest titer (day 12protein concentration).

FIG. 2 shows graphs indicating the viable cell density of GS-CHO cellsat day 0, Day 3 and Day 6 when exposed to increasing concentrations of2-hydroxybutyric acid (FIG. 2A), homocysteine (FIG. 2B) orindolecarboxylic acid (FIG. 2C). GS-CHO cells were inoculated in MediumA at low viable cell densities and were treated with reportedconcentrations of the inhibitors, individually. The effect of theinhibitors on growth of the cells was monitored for 6 days.

FIG. 3 shows the effect of increasing concentrations of two metabolites,4-hydroxyphenylpyruvate (FIG. 3A) and phenyllactate (FIG. 3B) on viablecell density of the GS-CHO cells. GS-CHO cells were inoculated in MediumA at low viable cell densities and were treated with reportedconcentrations of the inhibitors, individually.

FIGS. 4 and 5 show the effect of increasing concentrations of fourmetabolites, indolelactate (FIG. 4A), 3-(4-hydroxyphenyl)lactate (FIG.4B), sodium formate (FIG. 5A) and isovalerate (FIG. 5B), on viable celldensity of the GS-CHO cell. GS-CHO cells were inoculated in Medium A atlow viable cell densities and were treated with reported concentrationsof the inhibitors, individually.

FIG. 6 shows the effect of four metabolites (4-hydroxyphenylpyruvate,indolelactate, phenyllactate, and 3-(4-hydroxyphenyl)lactate) on viablecell density of GS-CHO cells as compared to a control where cells arecultured in the absence of the metabolites. GS-CHO cells were inoculatedin Medium A at low viable cell densities and were treated with freshmedia alone or fresh media comprising the above mentioned fourmetabolites in combination at concentrations that were detected on day 7of the HiPDOG culture (See Table 4). The effect of the inhibitors ongrowth of the cells was monitored for 6 days.

FIG. 7A shows the viable cell densities of GS-CHO cells in ‘HiPDOG’process (closed squares) and ‘Low AA+HiPDOG’ (closed diamonds) process.

FIG. 7B shows the culture titer (IgG) at different days in ‘HiPDOG’process (closed squares) and ‘Low AA+HiPDOG’ (closed diamonds) process.

FIGS. 8 and 9 show the amino acid concentrations of four amino acids in‘amino acid restricted HiPDOG process (Low AA+HiPDOG)’ (closed squares)and the ‘HiPDOG’ process (closed diamonds). The four amino acids includetyrosine (FIG. 7A), tryptophan (FIG. 8B), phenylalanine (FIG. 9A), andmethionine (FIG. 9B).

FIG. 10A shows the viable cell densities of GS-CHO cells during a cellculture process using different conditions disclosed in Example 5(HiPDOG1 (closed squares), HiPDOG2 (closed circles), Low 4AA+HipDOG(closed diamonds), Low 8AA+HipDOG (closed triangles)).

FIG. 10B shows the culture titer (IgG) at different days in a cellculture process using GS-CHO cells and different cell culture conditionsdisclosed in Example 5 ((HiPDOG1 (closed squares), HiPDOG2 (closedcircles), Low 4AA+HipDOG (closed diamonds), Low 8AA+HipDOG (closedtriangles)).

FIGS. 11, 12, 13 and 14 show the concentrations of tyrosine (FIG. 11A),methionine (FIG. 11B), phenylalanine (FIG. 12A), tryptophan (FIG. 12B),leucine (FIG. 13A), threonine (FIG. 13B), glycine (FIG. 14A) and serine(FIG. 14B) during the cell culture of GS-CHO cells using conditionsdisclosed in Example 5 ((HiPDOG1 (closed squares), (HiPDOG2 (closedcircles), Low 4AA+HipDOG (closed diamonds), Low 8AA+HipDOG (closedtriangles)).

FIGS. 15 and 16 show the concentration of 3-(4-hydroxyphenyl)lactate(FIG. 15A), isovalerate (FIG. 15B) and indole-3-lactate (FIG. 16) at day5, day 7 and day 9 of the cell culture of GS-CHO cells using conditionsdisclosed in Example 5 ((HiPDOG1 (closed squares), HiPDOG2 (closedcircles), Low 4AA+HipDOG (closed diamonds), Low 8AA+HipDOG (closedtriangles)).

FIG. 17 shows the viable cell densities of GS-CHO cells and culturetiter (IgG) during a cell culture process using conditions disclosed inExample 5 ((Low 4AA+HiPDOG (closed diamonds), Low 8AA+HipDOG (closedtriangles)).

FIGS. 18A and 18B show the viable cell densities of GS-CHO cells andculture titer (IgG) during a cell culture process using conditionsdisclosed in Example 6 ((HiPDOG (closed squares), Low 8AA+HipDOG (closedtriangles)).

FIGS. 19 and 20 show the concentration of 3-(4-hydroxyphenyl)lactate(FIG. 19A), isovalerate (FIG. 19B) and indole-3-lactate (FIG. 20) at day5, day 7 and day 10 of the cell culture of GS-CHO cells using conditionsdisclosed in Example 6 ((HiPDOG (closed squares), Low 8AA+HipDOG (closedtriangles)).

FIGS. 21A and 21B show the viable cell densities of GS-CHO cells andculture titer (IgG) during a cell culture process using differentconditions disclosed in Example 6 ((HiPDOG (closed squares), Low4AA+HipDOG (closed diamonds)).

FIGS. 22 and 23 show the concentration of 3-(4-hydroxyphenyl)lactate(FIG. 22A), isovalerate (FIG. 22B) and indole-3-lactate (FIG. 23) at day5 and day 7 of the cell culture of GS-CHO cells using conditionsdisclosed in Example 6 ((HiPDOG (closed squares), Low 4AA+HipDOG (closeddiamonds)).

DETAILED DESCRIPTION

The present invention provides methods and media for cell culture. Thepresent invention provides cell culture methods where the concentrationof at least one metabolite selected from 3-(4-hydroxyphenyl)lactate,4-hydroxyphenylpyruvate, phenyllactate, indolelactate, indolecarboxylicacid, homocysteine, 2-hydroxybutyric acid, isovalerate and formate,and/or at least one amino acid selected from phenylalanine, tyrosine,tryptophan, methionine, leucine, serine, threonine and glycine ismaintained at low levels in the cell culture medium. The inventors haveunexpectedly discovered that, in cell culture, and in particular in highdensity cell culture, such as for example fed-batch cell culture aimingat producing high amount of a recombinant protein of interest, thegrowth of cells were inhibited by the accumulation of metabolites suchas 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate,indolelactate, indolecarboxylic acid, homocysteine, 2-hydroxybutyricacid, isovalerate and formate in the cell culture medium. The inhibitoryeffect of these metabolites can be limited by maintaining theirconcentration in the cell culture medium below levels where they inhibitcell growth.

Methods Comprising Controlling the Metabolite Concentration in the CellCulture Medium at Low Levels

In some embodiments, the invention relates to a method of cell culturecomprising

(i) providing cells in a cell culture medium to start a cell cultureprocess, and,(ii) maintaining at least one metabolite selected from3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate,indolelactate, indolecarboxylic acid, homocysteine, 2-hydroxybutyricacid, isovalerate and formate below a concentration C1 in the cellculture medium, wherein C1 is 3 mM.

In some embodiments, C1 is 2 mM, 1.5 mM, 1 mM, 0.9 mM, 0.8 mM, 0.7 mM,0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM or 0.1 mM. In some embodiments,C1 is 1 mM. In some embodiments, C1 is 0.5 mM.

Methods Comprising Measuring the Metabolite Concentration in the CellCulture Medium

In some embodiments, step (ii) comprises the step of measuring theconcentration of said at least one metabolite. The concentration ofmetabolite can be measured by any method known to the skilled person,including off line and on line measurement methods.

The concentration of metabolites can be measured once or several timesduring the cell culture. In some embodiments, the metaboliteconcentration is measured continuously, intermittently, every 30 min,every hour, every two hours, twice a day, daily, or every two days. In apreferred embodiment the concentration of metabolite is measured daily.

An off line measurement method as used herein refers to a method wherethe measurement of a parameter such as a concentration is not automatedand integrated to the cell culture method. For example, a measurementmethod where a sample is manually taken from the cell culture medium sothat a specific concentration can be measured in said sample isconsidered as an off line measurement method.

Online measurement methods as used herein refer to methods where themeasurement of a parameter, such as a concentration, is automated andintegrated to the cell culture method. For example, a method using theRaman spectroscopy as disclosed in Example 7 is an on-line measurementmethod. Alternatively, the use of High Performance Liquid Chromatography(HPLC) or Ultra Performance Liquid Chromatography (UPLC) basedtechnology with an auto-sampler that draws samples from reactor andtransfers them to the equipment in a programmed manner is an onlinemeasurement method.

The concentration of metabolites can be measured by any method known tothe skilled person. Preferred methods to measure the concentration ofmetabolites in online or offline methods include for example LiquidChromatography such as High-Performance Liquid Chromatography (HPLC),Ultra Performance Liquid Chromatography (UPLC) or LiquidChromatography-Mass Spectrometry (LCMS), Nuclear Magnetic Resonance(NMR) or Gas Chromatography-Mass Spectrometry (GCMS).

In some embodiments, the concentration of metabolite is measured offline by taking a sample of the cell culture medium and measuring theconcentration of said at least one metabolite in said sample. In someembodiments, the concentration of metabolites is measured as disclosedin Example 2. A preferred method to measure the concentration ofmetabolites in an offline method is LCMS.

In some embodiments, the concentration of metabolite is measuredon-line. In some embodiments, the concentration of metabolite ismeasured online using Raman spectroscopy. In some embodiments, theconcentration of metabolite is measured on-line using Raman spectroscopyas disclosed in Example 7. In some embodiments, the concentration ofmetabolite is measured online using HPLC or UPLC based technology withan auto-sampler that draws samples from reactor and transfers them tothe equipment in a programmed manner.

In some embodiments, when the measured concentration is above apredefined value, the concentration of precursor of said at least onemetabolite in the cell culture medium is decreased. Said predefinedvalue is selected so that the decrease of concentration of saidprecursor prevents the concentration of metabolite to rise above C1. Thepredefined value can be equal C1 or can be a percentage of C1. In someembodiments the percentage is 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%of C1. In some embodiments the percentage is 80% of C1.

In some embodiments, when the measured concentration of3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate and/or phenyllactateis above said predefined value, the concentration of phenylalanine isdecreased in the cell culture medium.

In some embodiments, when the measured concentration of3-(4-hydroxyphenyl)lactate and/or 4-hydroxyphenylpyruvate is above saidpredefined value, the concentration of tyrosine is decreased in the cellculture medium.

In some embodiments, when the measured concentration of3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate and/or phenyllactateis above said predefined value, the concentrations of tyrosine andphenylalanine are decreased in the cell culture medium.

In some embodiments, when the measured concentration of indolelactateand/or indolecarboxylic acid is above said predefined value, theconcentration of tryptophan is decreased in the cell culture medium.

In some embodiments, when the measured concentration of homocysteineand/or 2-hydroxybutyric acid is above said predefined value, theconcentration of methionine is decreased in the cell culture medium.

In some embodiments, when the measured concentration of isovalerate isabove said predefined value, the concentration of leucine is decreasedin the cell culture medium.

In some embodiments, when the measured concentration of formate is abovesaid predefined value, the concentration of serine, threonine and/orglycine is decreased in the cell culture medium.

In some embodiments, when the measured concentration of formate is abovesaid predefined value, the concentration of serine is decreased in thecell culture medium.

In some embodiments, when the measured concentration of formate is abovesaid predefined value, the concentration of threonine is decreased inthe cell culture medium.

In some embodiments, when the measured concentration of formate is abovesaid predefined value, the concentration of glycine is decreased in thecell culture medium.

The concentration of precursor in the cell culture medium can bedecreased by reducing the amount of precursor provided to the cells, forexample by reducing the concentration of said precursor in the feedmedium, reducing the feed rate, or reducing the number or volume offeeds. For example, the feed medium can be replaced by a feed mediumcomprising a lower concentration of precursor.

In some embodiments of the above disclosed methods, step (ii) comprisesmaintaining 1, 2, 3, 4, 5, 6, 7, 8 or 9 of 3-(4-hydroxyphenyl)lactate,4-hydroxyphenylpyruvate, phenyllactate, indolelactate, indolecarboxylicacid, homocysteine, 2-hydroxybutyric acid, isovalerate and formate belowC1 in the cell culture medium.

In some embodiments of the above disclosed methods, step (ii) comprisesmaintaining 1, 2, 3, 4, 5, 6, or 7 of 3-(4-hydroxyphenyl)lactate,4-hydroxyphenylpyruvate, phenyllactate, indolelactate, indolecarboxylicacid, homocysteine and 2-hydroxybutyric acid below C1 in the cellculture medium.

In some embodiments of the above disclosed methods, step (ii) comprisesmaintaining 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate andphenyllactate below C1 in the cell culture medium.

In some embodiments of the above disclosed methods, step (ii) comprisesmaintaining indolelactate and indolecarboxylic acid below C1 in the cellculture medium.

In some embodiments of the above disclosed methods, step (ii) comprisesmaintaining homocysteine and 2-hydroxybutyric acid below C1 in the cellculture medium.

In some embodiments of the above disclosed methods, step (ii) comprisesmaintaining isovalerate below C1 in the cell culture medium.

In some embodiments of the above disclosed methods, step (ii) comprisesmaintaining formate below C1 in the cell culture medium.

In some embodiments of the above disclosed methods, step (ii) comprisesmaintaining isovalerate and 4-hydroxyphenylpyruvate below C1 in the cellculture medium.

In some embodiments of the above disclosed methods, step (ii) comprisesmaintaining 3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate,phenyllactate, indolelactate, indolecarboxylic acid, homocysteine,2-hydroxybutyric acid isovalerate and formate below C1 in the cellculture medium.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of 3-(4-hydroxyphenyl)lactate is maintained below 0.5 mM,0.4 mM, 0.3 mM, 0.2 mM, or 0.1 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of 3-(4-hydroxyphenyl)lactate is maintained below 0.3 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of 3-(4-hydroxyphenyl)lactate is maintained below 0.1 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of 4-hydroxyphenylpyruvate is maintained below 0.1 mM,0.08 mM, 0.06 mM, 0.05 mM, 0.04 mM, 0.03 mM or 0.02 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of 4-hydroxyphenylpyruvate is maintained below 0.05 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of 4-hydroxyphenylpyruvate is maintained below 0.02 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of phenyllactate is maintained below 0.5 mM, 0.4 mM, 0.3mM, 0.2 mM, or 0.1 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of phenyllactate is maintained below 0.2 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of phenyllactate is maintained below 0.1 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of indolelactate is maintained below 3 mM, 2 mM, 1 mM, 0.5mM, 0.3 mM or 0.1 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of indolelactate is maintained below 1 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of indolelactate is maintained below 0.3 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of indolelactate is maintained below 0.1 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of indolecarboxylic acid is maintained below 1 mM, 0.8 mM,0.6 mM, 0.4 mM or 0.2 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of indolecarboxylic acid is maintained below 0.5 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of indolecarboxylic acid is maintained below 0.2 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of homocysteine is maintained below 0.5 mM, 0.4 mM, 0.3mM, 0.2 mM, or 0.1 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of homocysteine is maintained below 0.3 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of homocysteine is maintained below 0.1 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of 2-hydroxybutyric acid is maintained below 1 mM, 0.8 mM,0.6 mM, 0.4 mM or 0.2 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of 2-hydroxybutyric acid is maintained below 0.5 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of 2-hydroxybutyric acid is maintained below 0.2 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of isovalerate is maintained below 2 mM, 1 mM, 0.8 mM, 0.6mM, 0.4 mM or 0.2 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of isovalerate is maintained below 1 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of isovalerate is maintained below 0.5 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of formate is maintained below 4 mM, 3 mM, 2 mM, 1 mM, 0.5mM or 0.2 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of formate is maintained below 3 mM.

In some embodiments of the above disclosed methods, in step (ii), theconcentration of formate is maintained below 1 mM.

Methods Comprising Controlling the Amino Acid Concentration in the CellCulture Medium at Low Levels

In some embodiments, the invention relates to a method of cell culturecomprising

(i) providing cells in a cell culture medium to start a cell cultureprocess, and,(ii) maintaining at least one amino acid selected from phenylalanine,tyrosine, tryptophan, methionine, leucine, serine, threonine and glycinebelow a concentration C2 in the cell culture medium, wherein C2 is 2 mM.

In some embodiments, said concentration is maintained between 0.1 mM andC2, 0.2 mM and C2, 0.3 mM and C2, 0.4 mM and C2, or 0.5 mM and C2. Insome embodiments, said concentration is maintained between 0.5 mM andC2.

In some embodiments, C2 is 2 mM, 1.5 mM, 1 mM, 0.9 mM, 0.8 mM, 0.7 mM,0.6 mM. In some embodiments, C2 is 1 mM.

Methods Comprising Measuring the Amino Acid Concentration in the CellCulture Medium

In some embodiments, step (ii) comprises the step of measuring theconcentration of said at least one amino acid. The concentration ofamino acid can be measured by any method known to the skilled person,including off line and on line measurement methods.

The concentration of amino acids can be measured once or several timesduring the cell culture. In some embodiments, the amino acidconcentration is measured continuously, intermittently, every 30 min,every hour, every two hours, twice a day, daily, or every two days. In apreferred embodiment, the concentration of amino acid is measured daily.

The concentration of amino acid can be measured by any method known tothe skilled person. Preferred methods to measure the concentration ofamino acids in online or offline methods include for example LiquidChromatography such HPLC, UPLC or LCMS, NMR or GCMS.

In some embodiments, the concentration of amino acid is measured offline by taking a sample of the cell culture medium and measuring theconcentration of said at least one amino acid in said sample. In someembodiments, the concentration of amino acid is measured as disclosed inExample 4. A preferred method to measure the concentration of aminoacids in an off line method is UPLC.

In some embodiments, the concentration of amino acid is measured online.In some embodiments, the concentration of amino acid is measured on-lineusing Raman spectroscopy. In some embodiments, the concentration ofamino acid is measured on-line using Raman spectroscopy as disclosed inExample 7. In some embodiments, the concentration of amino acid ismeasured online using HPLC or UPLC based technology with an auto-samplerthat draws sample from reactor and transfers to the equipment in aprogrammed manner.

In some embodiments, when the measured concentration is above apredefined value, the concentration of said at least one amino acid inthe cell culture medium is decreased. The predefined value can be equalC2 or can be a percentage of C2. In some embodiments the percentage is50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% of C2. In some embodiments thepercentage is 80% of C2.

The concentration of amino acid in the cell culture medium can bedecreased by reducing the amount of amino acid provided to the cells,for example by reducing the concentration of said amino acid in the feedmedium, reducing the feed rate, or reducing the number or volume offeeds. For example, the feed medium can be replaced by a feed mediumcomprising a lower concentration of amino acid.

Concentration of Phenylalanine, Tyrosine, Tryptophan Methionine,Leucine, Serine, Threonine and Glycine in the Cell Culture Medium

In some embodiments of any of the above disclosed methods, in step (ii),the concentration of phenylalanine is maintained below 2 mM in the cellculture medium. In a preferred embodiment, the concentration ofphenylalanine is maintained between 0.1 and 2 mM in the cell culturemedium. In a preferred embodiment, the concentration of phenylalanine ismaintained between 0.1 and 1 mM in the cell culture medium. In apreferred embodiment, the concentration of phenylalanine is maintainedbetween 0.2 and 1 mM in the cell culture medium. In a preferredembodiment, the concentration of phenylalanine is maintained between 0.5and 1 mM in the cell culture medium.

In some embodiments of any of the above disclosed methods, in step (ii),the concentration of tyrosine is maintained below 2 mM in the cellculture medium. In a preferred embodiment, the concentration of tyrosineis maintained between 0.1 and 2 mM in the cell culture medium. In apreferred embodiment, the concentration of tyrosine is maintainedbetween 0.1 and 1 mM in the cell culture medium. In a preferredembodiment, the concentration of tyrosine is maintained between 0.2 and1 mM in the cell culture medium. In a preferred embodiment, theconcentration of tyrosine is maintained between 0.5 and 1 mM in the cellculture medium.

In some embodiments of any of the above disclosed methods, in step (ii),the concentration of tryptophan is maintained below 2 mM in the cellculture medium. In a preferred embodiment, the concentration oftryptophan is maintained between 0.1 and 2 mM in the cell culturemedium. In a preferred embodiment, the concentration of tryptophan ismaintained between 0.1 and 1 mM in the cell culture medium. In apreferred embodiment, the concentration of tryptophan is maintainedbetween 0.2 and 1 mM in the cell culture medium. In a preferredembodiment, the concentration of tryptophan is maintained between 0.5and 1 mM in the cell culture medium.

In some embodiments of any of the above disclosed methods, in step (ii),the concentration of methionine is maintained below 2 mM in the cellculture medium. In a preferred embodiment, the concentration ofmethionine is maintained between 0.1 and 2 mM in the cell culturemedium. In a preferred embodiment, the concentration of methionine ismaintained between 0.1 and 1 mM in the cell culture medium. In apreferred embodiment, the concentration of methionine is maintainedbetween 0.2 and 1 mM in the cell culture medium. In a preferredembodiment, the concentration of methionine is maintained between 0.5and 1 mM in the cell culture medium.

In some embodiments of any of the above disclosed methods, in step (ii),the concentration of leucine is maintained below 2 mM in the cellculture medium. In a preferred embodiment, the concentration of leucineis maintained between 0.1 and 2 mM in the cell culture medium. In apreferred embodiment, the concentration of leucine is maintained between0.1 and 1 mM in the cell culture medium. In a preferred embodiment, theconcentration of leucine is maintained between 0.2 and 1 mM in the cellculture medium. In a preferred embodiment, the concentration of leucineis maintained between 0.5 and 1 mM in the cell culture medium.

In some embodiments of any of the above disclosed methods, in step (ii),the concentration of serine is maintained below 2 mM in the cell culturemedium. In a preferred embodiment, the concentration of serine ismaintained between 0.1 and 2 mM in the cell culture medium. In apreferred embodiment, the concentration of serine is maintained between0.1 and 1 mM in the cell culture medium. In a preferred embodiment, theconcentration of serine is maintained between 0.2 and 1 mM in the cellculture medium. In a preferred embodiment, the concentration of serineis maintained between 0.5 and 1 mM in the cell culture medium.

In some embodiments of any of the above disclosed methods, in step (ii),the concentration of threonine is maintained below 2 mM in the cellculture medium. In a preferred embodiment, the concentration ofthreonine is maintained between 0.1 and 2 mM in the cell culture medium.In a preferred embodiment, the concentration of threonine is maintainedbetween 0.1 and 1 mM in the cell culture medium. In a preferredembodiment, the concentration of threonine is maintained between 0.2 and1 mM in the cell culture medium. In a preferred embodiment, theconcentration of threonine is maintained between 0.5 and 1 mM in thecell culture medium.

In some embodiments of any of the above disclosed methods, in step (ii),the concentration of glycine is maintained below 2 mM in the cellculture medium. In a preferred embodiment, the concentration of glycineis maintained between 0.1 and 2 mM in the cell culture medium. In apreferred embodiment, the concentration of glycine is maintained between0.1 and 1 mM in the cell culture medium. In a preferred embodiment, theconcentration of glycine is maintained between 0.2 and 1 mM in the cellculture medium. In a preferred embodiment, the concentration of glycineis maintained between 0.5 and 1 mM in the cell culture medium.

In a preferred embodiment of any of the above disclosed methods, in step(ii), the concentration of tyrosine, phenylalanine and leucine ismaintained below 2 mM, preferably between 0.1 and 2 mM, between 0.1 and1 mM, between 0.2 and 1 mM or between 0.5 and 1 mM in the cell culturemedium.

In some embodiments, the cell culture medium comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12 or 13 of glycine, valine, leucine, isoleucine,proline, serine, threonine, lysine, arginine, histidine, aspartate,glutamate and asparagine at a concentration above 1 mM, 1.5 mM, 2 mM, 3mM or 5 mM.

In some embodiments, the cell culture medium comprises one of glycine,valine, leucine, isoleucine, proline, serine, threonine, lysine,arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 2 mM.

In some embodiments, the cell culture medium comprises one of glycine,valine, leucine, isoleucine, proline, serine, threonine, lysine,arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 5 mM.

In some embodiments, the cell culture medium comprises 1, 2, 3, 4, 5, 6,7, 8 or 9, of valine, isoleucine, proline, lysine, arginine, histidine,aspartate, glutamate and asparagine at a concentration above 1 mM, 1.5mM, 2 mM, 3 mM or 5 mM.

In some embodiments, the cell culture medium comprises one of valine,isoleucine, proline, lysine, arginine, histidine, aspartate, glutamateand asparagine at a concentration above 2 mM.

In some embodiments, the cell culture medium comprises one of valine,isoleucine, proline, lysine, arginine, histidine, aspartate, glutamateand asparagine at a concentration above 5 mM.

Concentration of Lactate and Ammonia

In some embodiments of any of the above disclosed methods, othermetabolites inhibiting growth of cells, such as lactate and ammonia arealso maintained at low levels in the cell culture medium. Methods tokeep lactate and ammonia at low levels are known to the skilled person.

For example, lactate can be kept at low levels in cell culture by usingmethods disclosed in WO2004104186, Gagnon et al, Biotechnology andBioengineering, Vol. 108, No. 6, June, 2011 (Gagnon et Al) orWO2004048556.

Various other strategies can be employed to restrict lactate productionand/or induce lactate consumption. These include culturing cells underslightly reduced pH (6.7-7.0), culturing cells at low glucoseconcentrations by using alternative carbon sources including but notlimited to fructose (Wlaschin & Hu, 2007) and galactose (Altamirano etal, 2006), using a cell line that has reduced protein levels ofglycolytic enzymes including but not limited to hexose transporter orlactate dehydrogenase (Kim & Lee, 2007a), employing a cell line withsuppressed cellular protein levels of both lactate dehydrogenase andpyruvate dehydrogenase kinase (Zhou et al, 2011), or cell line withover-expression of pyruvate carboxylase enzyme (Kim & Lee, 2007b), orwith the use of inhibitors (small molecule or protein based) forsignaling pathways (such as AKT (Mulukutla et al, 2012), mTOR (Duvel etal, 2010; Lee & Lee, 2012), HIF1a) that regulate the activity of energymetabolism pathways (glycolysis, TCA cycle, and redox pathway).

In a preferred embodiment, lactate is maintained at low levels by usingthe high-end pH-controlled delivery of glucose (HIPDOG process)disclosed in Gagnon et al.

In some embodiments of any of the above disclosed methods lactate ismaintained at low levels in the cell culture medium. In a preferredembodiment, concentration of lactate in the cell culture medium ismaintained below 90 mM. In a preferred embodiment, the concentration oflactate in the cell culture medium is maintained below 70 mM. In apreferred embodiment, the concentration of lactate in the cell culturemedium is maintained below 50 mM. In a preferred embodiment, theconcentration of lactate in the cell culture medium is maintained below40 mM. In a preferred embodiment, lactate is maintained at low levels bycontrolling the amount of glucose provided to the cell culture. In apreferred embodiment, lactate is maintained at low levels by using theHIPDOG process. In a preferred embodiment, a pH sensor is used tomonitor pH of the cell culture, and, in response to a rise above apredetermined pH value, glucose is fed to the cell culture. In apreferred embodiment, the predetermined pH value is approximately 7.

Ammonia can be kept at low levels in cell culture by any method known tothe skilled person such as for example the methods disclosed in Butleret Al, Cytotechnology 15: 87-94, 1994 or Hong et Al, Appl MicrobiolBiotechnol (2010) 88:869-876. Alternatively, ammonia can be kept at lowlevels by using a glutamine synthetase (GS) expression system. Suchsystems are commercially available (Lonza) and can be used to generaterecombinant cell lines. Cell lines using GS expression systemdemonstrate the gain of function to synthesize glutamine in vivo,thereby completely relieving cellular dependence on the externallysupplied glutamine. Since the major fraction of ammonia produced inculture is from the catabolysis of externally supplied glutamine, such again of metabolic function reduces the levels of ammonia produced inculture.

In some embodiments of any of the above disclosed methods, ammonia ismaintained at low levels in the cell culture medium. In a preferredembodiment, concentration of ammonia in the cell culture medium ismaintained below 20 mM. In a preferred embodiment, the concentration ofammonia in the cell culture medium is maintained below 10 mM. In apreferred embodiment, the concentration of ammonia in the cell culturemedium is maintained below 8 mM.

Cells

Any cell susceptible to cell culture may be utilized in accordance withthe present invention. In some embodiments, the cell is a mammaliancell. Non-limiting examples of mammalian cells that may be used inaccordance with the present invention include BALB/c mouse myeloma line(NSO/I, ECACC No: 85110503); human retinoblasts (PER.C6, CruCell,Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells +/−DHFR (CHO, Urlaub and Chasin, Proc. Natl.Acad. Sci. USA, 77:4216, 1980); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1 587); humancervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2). In some preferred embodiment, the cellsare CHO cells. In some preferred embodiments, the cells are GS-cells.

Additionally, any number of commercially and non-commercially availablehybridoma cell lines may be utilized in accordance with the presentinvention. The term “hybridoma” as used herein refers to a cell orprogeny of a cell resulting from fusion of an immortalized cell and anantibody-producing cell. Such a resulting hybridoma is an immortalizedcell that produces antibodies. Individual cells used to create thehybridoma can be from any mammalian source, including, but not limitedto, rat, pig, rabbit, sheep, pig, goat, and human. In some embodiments,a hybridoma is a trioma cell line, which results when progeny ofheterohybrid myeloma fusions, which are the product of a fusion betweenhuman cells and a murine myeloma cell line, are subsequently fused witha plasma cell. In some embodiments, a hybridoma is any immortalizedhybrid cell line that produces antibodies such as, for example,quadromas (See, e.g., Milstein et al., Nature, 537:3053, 1983). Oneskilled in the art will appreciate that hybridoma cell lines might havedifferent nutrition requirements and/or might require different cultureconditions for optimal growth, and will be able to modify conditions asneeded.

Cell Culture Methods

The terms “culture” and “cell culture” as used herein refer to a cellpopulation that is suspended in a medium under conditions suitable tosurvival and/or growth of the cell population. As will be clear to thoseof ordinary skill in the art, in some embodiments, these terms as usedherein refer to the combination comprising the cell population and themedium in which the population is suspended. In some embodiments, thecells of the cell culture comprise mammalian cells.

The present invention may be used with any cell culture method that isamenable to the desired process (e.g., production of a recombinantprotein (e.g., antibody)). As a non-limiting example, cells may be grownin batch or fed-batch cultures, where the culture is terminated aftersufficient expression of the recombinant protein (e.g., antibody), afterwhich the expressed protein (e.g., antibody) is harvested.Alternatively, as another non-limiting example, cells may be grown inbatch-refeed, where the culture is not terminated and new nutrients andother components are periodically or continuously added to the culture,during which the expressed recombinant protein (e.g., antibody) isharvested periodically or continuously. Other suitable methods (e.g.,spin-tube cultures) are known in the art and can be used to practice thepresent invention.

In some embodiments, a cell culture suitable for the present inventionis a fed-batch culture. The term “fed-batch culture” as used hereinrefers to a method of culturing cells in which additional components areprovided to the culture at a time or times subsequent to the beginningof the culture process. Such provided components typically comprisenutritional components for the cells which have been depleted during theculturing process. A fed-batch culture is typically stopped at somepoint and the cells and/or components in the medium are harvested andoptionally purified. In some embodiments, the fed-batch culturecomprises a base medium supplemented with feed media.

Cells may be grown in any convenient volume chosen by the practitioner.For example, cells may be grown in small scale reaction vessels rangingin volume from a few milliliters to several liters. Alternatively, cellsmay be grown in large scale commercial Bioreactors ranging in volumefrom approximately at least 1 liter to 10, 50, 100, 250, 500, 1000,2500, 5000, 8000, 10,000, 12,000, 15000, 20000 or 25000 liters or more,or any volume in between.

The temperature of a cell culture will be selected based primarily onthe range of temperatures at which the cell culture remains viable andthe range in which a high level of desired product (e.g., a recombinantprotein) is produced. In general, most mammalian cells grow well and canproduce desired products (e.g., recombinant proteins) within a range ofabout 25° C. to 42° C., although methods taught by the presentdisclosure are not limited to these temperatures. Certain mammaliancells grow well and can produce desired products (e.g., recombinantproteins or antibodies) within the range of about 35° C. to 40° C. Incertain embodiments, a cell culture is grown at a temperature of 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44 or 45° C. at one or more times during the cellculture process. Those of ordinary skill in the art will be able toselect appropriate temperature or temperatures in which to grow cells,depending on the particular needs of the cells and the particularproduction requirements of the practitioner. The cells may be grown forany amount of time, depending on the needs of the practitioner and therequirement of the cells themselves. In some embodiment, the cells aregrown at 37° C. In some embodiments, the cells are grown at 36.5° C.

In some embodiments, the cells may be grown during the initial growthphase (or growth phase) for a greater or lesser amount of time,depending on the needs of the practitioner and the requirement of thecells themselves. In some embodiments, the cells are grown for a periodof time sufficient to achieve a predefined cell density. In someembodiments, the cells are grown for a period of time sufficient toachieve a cell density that is a given percentage of the maximal celldensity that the cells would eventually reach if allowed to growundisturbed. For example, the cells may be grown for a period of timesufficient to achieve a desired viable cell density of 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percentof maximal cell density. In some embodiments, the cells are grown untilthe cell density does not increase by more than 15%, 14%, 13%, 12%, 11%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% per day of culture. In someembodiments, the cells are grown until the cell density does notincrease by more than 5% per day of culture.

In some embodiment the cells are allowed to grow for a defined period oftime. For example, depending on the starting concentration of the cellculture, the temperature at which the cells are grown, and the intrinsicgrowth rate of the cells, the cells may be grown for 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days,preferably for 4 to 10 days. In some cases, the cells may be allowed togrow for a month or more. The practitioner of the present invention willbe able to choose the duration of the initial growth phase depending onprotein production requirements and the needs of the cells themselves.

The cell culture may be agitated or shaken during the initial culturephase in order to increase oxygenation and dispersion of nutrients tothe cells. In accordance with the present invention, one of ordinaryskill in the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor during theinitial growth phase, including but not limited to pH, temperature,oxygenation, etc.

At the end of the initial growth phase, at least one of the cultureconditions may be shifted so that a second set of culture conditions isapplied and a metabolic shift occurs in the culture. A metabolic shiftcan be accomplished by, e.g., a change in the temperature, pH,osmolality or chemical inductant level of the cell culture. In onenon-limiting embodiment, the culture conditions are shifted by shiftingthe temperature of the culture. However, as is known in the art,shifting temperature is not the only mechanism through which anappropriate metabolic shift can be achieved. For example, such ametabolic shift can also be achieved by shifting other cultureconditions including, but not limited to, pH, osmolality, and sodiumbutyrate levels. The timing of the culture shift will be determined bythe practitioner of the present invention, based on protein productionrequirements or the needs of the cells themselves.

When shifting the temperature of the culture, the temperature shift maybe relatively gradual. For example, it may take several hours or days tocomplete the temperature change. Alternatively, the temperature shiftmay be relatively abrupt. For example, the temperature change may becomplete in less than several hours. Given the appropriate productionand control equipment, such as is standard in the commercial large-scaleproduction of polypeptides or proteins, the temperature change may evenbe complete within less than an hour.

In some embodiments, once the conditions of the cell culture have beenshifted as discussed above, the cell culture is maintained for asubsequent production phase under a second set of culture conditionsconducive to the survival and viability of the cell culture andappropriate for expression of the desired polypeptide or protein atcommercially adequate levels.

As discussed above, the culture may be shifted by shifting one or moreof a number of culture conditions including, but not limited to,temperature, pH, osmolality, and sodium butyrate levels. In someembodiments, the temperature of the culture is shifted. According tothis embodiment, during the subsequent production phase, the culture ismaintained at a temperature or temperature range that is lower than thetemperature or temperature range of the initial growth phase. Asdiscussed above, multiple discrete temperature shifts may be employed toincrease cell density or viability or to increase expression of therecombinant protein.

In some embodiments, the cells may be maintained in the subsequentproduction phase until a desired cell density or production titer isreached. In another embodiment of the present invention, the cells areallowed to grow for a defined period of time during the subsequentproduction phase. For example, depending on the concentration of thecell culture at the start of the subsequent growth phase, thetemperature at which the cells are grown, and the intrinsic growth rateof the cells, the cells may be grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. In some cases, thecells may be allowed to grow for a month or more. The practitioner ofthe present invention will be able to choose the duration of thesubsequent production phase depending on polypeptide or proteinproduction requirements and the needs of the cells themselves.

The cell culture may be agitated or shaken during the subsequentproduction phase in order to increase oxygenation and dispersion ofnutrients to the cells. In accordance with the present invention, one ofordinary skill in the art will understand that it can be beneficial tocontrol or regulate certain internal conditions of the bioreactor duringthe subsequent growth phase, including but not limited to pH,temperature, oxygenation, etc.

In some embodiments, the cells express a recombinant protein and thecell culture method of the invention comprises a growth phase and aproduction phase.

In some embodiment step (ii) of any of the methods disclosed herein isapplied during the totality of the cell culture method. In someembodiment step (ii) of any of the methods disclosed herein is appliedduring a part of the cell culture method. In some embodiments, step (ii)is applied until a predetermined viable cell density is obtained.

In some embodiments, the cell culture method of the invention comprisesa growth phase and a production phase and step (ii) is applied duringthe growth phase. In some embodiments, the cell culture method of theinvention comprises a growth phase and a production phase and step (ii)is applied during a part of the growth phase. In some embodiments, thecell culture method of the invention comprises a growth phase and aproduction phase and step (ii) is applied during the growth phase andthe production phase. In step (ii) of any of the method disclosedherein, the term “maintaining” can refer to maintaining theconcentration of amino acid or metabolite below C1 or C2 for the entireculture process (until harvesting) or for a part of the culture processsuch as for example the growth phase, a part of the growth phase oruntil a predetermined cell density is obtained.

Improvement of Cell Growth and Productivity

In some embodiments of any of the above mentioned methods, cell growthand/or productivity are increased as compared to a control culture, saidcontrol culture being identical except that it does not comprise step(ii).

In some embodiments of any of the above mentioned methods, the method ofthe invention is a method for improving cell growth. In some embodiment,the method of the invention is a method for improving cell growth inhigh density cell culture at high cell density.

High cell density as used herein refers to cell density above 1×10⁶cells/mL, 5×10⁶ cells/mL, 1×10⁷ cells/mL, 5×10⁷ cells/mL, 1×10⁸ cells/mLor 5×10⁸ cells/mL, preferably above 1×10⁷ cells/mL, more preferablyabove 5×10⁷ cells/mL.

In some embodiments, the method of the invention is a method forimproving cell growth in a cell culture where cell density is above1×10⁶ cells/mL, 5×10⁶ cells/mL, 1×10⁷ cells/mL, 5×10⁷ cells/mL, 1×10⁸cells/mL or 5×10⁸ cells/mL. In some embodiments, the method of theinvention is a method for improving cell growth in a cell culture wheremaximum cell density is above 1×10⁶ cells/mL, 5×10⁶ cells/mL, 1×10⁷cells/mL, 5×10⁷ cells/mL, 1×10⁸ cells/mL or 5×10⁸ cells/mL.

In some embodiments, cell growth is determined by viable cell density(VCD), maximum viable cell density, or Integrated viable cell count(IVCC). In some embodiments, cell growth is determined by maximum viablecell density.

The term “viable cell density” as used herein refers to the number ofcells present in a given volume of medium. Viable cell density can bemeasured by any method known to the skilled person. Preferably, Viablecell density is measured using an automated cell counter such asBioprofile Flex®. The term maximum cell density as used herein refers tothe maximum cell density achieved during the cell culture. The term“cell viability” as used herein refers to the ability of cells inculture to survive under a given set of culture conditions orexperimental variations. Those of ordinary skill in the art willappreciate that one of many methods for determining cell viability areencompassed in this invention. For example, one may use a dye (e.g.,trypan blue) that does not pass through the membrane of a living cell,but can pass through the disrupted membrane of a dead or dying cell inorder to determine cell viability.

The term “Integrated viable cell count (IVCC)” as used herein refers toas the area under the viable cell density (VCD) curve. IVCC can becalculated using the following formula:

IVCC_(t+1)=IVCC_(t)+(VCD_(t)+VCD_(t+1))*(Δt)/2

where Δt is the time difference between t and t+1 time points.IVCC_(t=0) can be assumed negligible. VCD_(t) and VCD_(t+1) are viablecell densities at t and t+1 time points.

The term “titer” as used herein refers, for example, to the total amountof recombinantly expressed protein produced by a cell culture in a givenamount of medium volume. Titer is typically expressed in units of gramsof protein per liter of medium.

In some embodiments, cell growth is increased by at least 5%, 10%, 15%,20% or 25% as compared to the control culture. In some embodiments, cellgrowth is increased by at least 10% as compared to the control culture.In some embodiments, cell growth is increased by at least 20% ascompared to the control culture.

In some embodiments, the productivity is determined by titer and/orvolumetric productivity.

The term “titer” as used herein refers, for example, to the total amountof recombinantly expressed protein produced by a cell culture in a givenamount of medium volume. Titer is typically expressed in units of gramsof protein per liter of medium.

In some embodiments, the productivity is determined by titer. In someembodiments, the productivity is increased by at least 5%, 10%, 15%, 20%or 25% as compared to the control culture.

In some embodiments, the productivity is increased by at least 10% ascompared to a control culture.

In some embodiments, the productivity is increased by at least 20% ascompared to a control culture.

In some embodiments, the maximum cell density of the cell culture isgreater than 1×10⁶ cells/mL, 5×10⁶ cells/mL, 1×10⁷ cells/mL, 5×10⁷cells/mL, 1×10⁸ cells/mL or 5×10⁸ cells/mL. In some embodiments, themaximum cell density of the cell culture is greater than 5×10⁶ cells/mL.In some embodiments, the maximum cell density of the cell culture isgreater than 1×10⁸ cells/mL.

Cell Culture Media

The terms “medium”, “cell culture medium” and “culture medium” as usedherein refer to a solution containing nutrients which nourish growingmammalian cells. Typically, such solutions provide essential andnon-essential amino acids, vitamins, energy sources, lipids, and traceelements required by the cell for minimal growth and/or survival. Such asolution may also contain supplementary components that enhance growthand/or survival above the minimal rate, including, but not limited to,hormones and/or other growth factors, particular ions (such as sodium,chloride, calcium, magnesium, and phosphate), buffers, vitamins,nucleosides or nucleotides, trace elements (inorganic compounds usuallypresent at very low final concentrations), inorganic compounds presentat high final concentrations (e.g., iron), amino acids, lipids, and/orglucose or other energy source. In some embodiments, a medium isadvantageously formulated to a pH and salt concentration optimal forcell survival and proliferation. In some embodiments, a medium is a feedmedium that is added after the beginning of the cell culture.

A wide variety of mammalian growth media may be used in accordance withthe present invention. In some embodiments, cells may be grown in one ofa variety of chemically defined media, wherein the components of themedia are both known and controlled. In some embodiments, cells may begrown in a complex medium, in which not all components of the medium areknown and/or controlled.

Chemically defined growth media for mammalian cell culture have beenextensively developed and published over the last several decades. Allcomponents of defined media are well characterized, and so defined mediado not contain complex additives such as serum or hydrolysates. Earlymedia formulations were developed to permit cell growth and maintenanceof viability with little or no concern for protein production. Morerecently, media formulations have been developed with the expresspurpose of supporting highly productive recombinant protein producingcell cultures. Such media are preferred for use in the method of theinvention. Such media generally comprises high amounts of nutrients andin particular of amino acids to support the growth and/or themaintenance of cells at high density. If necessary, these media can bemodified by the skilled person for use in the method of the invention.For example, the skilled person may decrease the amount ofphenylalanine, tyrosine, tryptophan and/or methionine in these media fortheir use as base media or feed media in a method as disclosed herein.

Not all components of complex media are well characterized, and socomplex media may contain additives such as simple and/or complex carbonsources, simple and/or complex nitrogen sources, and serum, among otherthings. In some embodiments, complex media suitable for the presentinvention contains additives such as hydrolysates in addition to othercomponents of defined medium as described herein.

In some embodiments, defined media typically includes roughly fiftychemical entities at known concentrations in water. Most of them alsocontain one or more well-characterized proteins such as insulin, IGF-1,transferrin or BSA, but others require no protein components and so arereferred to as protein-free defined media. Typical chemical componentsof the media fall into five broad categories: amino acids, vitamins,inorganic salts, trace elements, and a miscellaneous category thatdefies neat categorization.

Cell culture medium may be optionally supplemented with supplementarycomponents. The term “supplementary components” as used herein refers tocomponents that enhance growth and/or survival above the minimal rate,including, but not limited to, hormones and/or other growth factors,particular ions (such as sodium, chloride, calcium, magnesium, andphosphate), buffers, vitamins, nucleosides or nucleotides, traceelements (inorganic compounds usually present at very low finalconcentrations), amino acids, lipids, and/or glucose or other energysource. In some embodiments, supplementary components may be added tothe initial cell culture. In some embodiments, supplementary componentsmay be added after the beginning of the cell culture.

Typically, trace elements refer to a variety of inorganic salts includedat micromolar or lower levels. For example, commonly included traceelements are zinc, selenium, copper, and others. In some embodiments,iron (ferrous or ferric salts) can be included as a trace element in theinitial cell culture medium at micromolar concentrations. Manganese isalso frequently included among the trace elements as a divalent cation(MnCl₂ or MnSO₄) in a range of nanomolar to micromolar concentrations.Numerous less common trace elements are usually added at nanomolarconcentrations.

In some embodiments, the medium used in the method of the invention is amedium suitable for supporting high cell density, such as for example1×10⁶ cells/mL, 5×10⁶ cells/mL, 1×10⁷ cells/mL, 5×10⁷ cells/mL, 1×10⁸cells/mL or 5×10⁸ cells/mL, in a cell culture. In some embodiments, thecell culture is a mammalian cell fed-batch culture, preferably a CHOcells fed-batch culture.

In some embodiments, the cell culture medium comprises phenylalanine ata concentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises tyrosine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises tryptophan at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises methionine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises leucine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises serine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises threonine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises glycine at aconcentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between 0.1to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises two ofphenylalanine, tyrosine, tryptophan, methionine, leucine, serine,threonine and glycine at a concentration below 2 mM, below 1 mM, between0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine andtyrosine at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine andtryptophan at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine andmethionine at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises tyrosine andtryptophan at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises tyrosine andmethionine at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises tryptophan andmethionine at a concentration below 2 mM, below 1 mM, between 0.1 and 2mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises three ofphenylalanine, tyrosine, tryptophan, methionine, leucine, serine,threonine and glycine at a concentration below 2 mM, below 1 mM, between0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine,tyrosine and tryptophan at a concentration below 2 mM, below 1 mM,between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM orbetween 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine,tyrosine and methionine at a concentration below 2 mM, below 1 mM,between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM orbetween 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine,tryptophan and methionine at a concentration below 2 mM, below 1 mM,between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM orbetween 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises tyrosine,tryptophan and methionine at a concentration below 2 mM, below 1 mM,between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM orbetween 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises four ofphenylalanine, tyrosine, tryptophan, methionine, leucine, serine,threonine and glycine at a concentration below 2 mM, below 1 mM, between0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine,tyrosine, tryptophan and methionine at a concentration below 2 mM, below1 mM, between 0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mMor between 0.5 to 1 mM.

In some embodiments, the cell culture medium comprises five ofphenylalanine, tyrosine, tryptophan, methionine, leucine, serine,threonine and glycine at a concentration below 2 mM, below 1 mM, between0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5to 1 mM.

In some embodiments, the cell culture medium comprises six ofphenylalanine, tyrosine, tryptophan, methionine, leucine, serine,threonine and glycine at a concentration below 2 mM, below 1 mM, between0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5to 1 mM.

In some embodiments, the cell culture medium comprises seven ofphenylalanine, tyrosine, tryptophan, methionine, leucine, serine,threonine and glycine at a concentration below 2 mM, below 1 mM, between0.1 and 2 mM, between 0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5to 1 mM.

In some embodiments, the cell culture medium comprises phenylalanine,tyrosine, tryptophan, methionine, leucine, serine, threonine and glycineat a concentration below 2 mM, below 1 mM, between 0.1 and 2 mM, between0.1 to 1 mM, between 0.5 and 1.5 mM or between 0.5 to 1 mM.

In some embodiments, the cell culture medium further comprises at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of glycine, valine, leucine,isoleucine, proline, serine, threonine, lysine, arginine, histidine,aspartate, glutamate and asparagine at a concentration above 2 mM, 3 mM,4 mM, 5 mM, 10 mM, 15 mM, preferably 2 mM.

In some embodiments, the cell culture medium further comprises at least5 of glycine, valine, leucine, isoleucine, proline, serine, threonine,lysine, arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2mM.

In some embodiments, the cell culture medium further comprises glycine,valine, leucine, isoleucine, proline, serine, threonine, lysine,arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2mM.

In some embodiments, the cell culture medium further comprises at least1, 2, 3, 4, 5, 6, 7, 8, or 9 of valine, isoleucine, proline, lysine,arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2mM.

In some embodiments, the cell culture medium further comprises at least5 of valine, isoleucine, proline, lysine, arginine, histidine,aspartate, glutamate and asparagine at a concentration above 2 mM, 3 mM,4 mM, 5 mM, 10 mM, 15 mM, preferably 2 mM.

In some embodiments, the cell culture medium further comprises valine,isoleucine, proline, lysine, arginine, histidine, aspartate, glutamateand asparagine at a concentration above 2 mM, 3 mM, 4 mM, 5 mM, 10 mM,15 mM, preferably 2 mM.

In some embodiments, the cell culture medium comprises serine at aconcentration above 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably10 mM.

In some embodiments, the cell culture medium comprises valine at aconcentration above 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably10 mM.

In some embodiments, the cell culture medium comprises cysteine at aconcentration above 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably10 mM.

In some embodiments, the cell culture medium comprises isoleucine at aconcentration above 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably10 mM.

In some embodiments, the cell culture medium comprises leucine at aconcentration above 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably10 mM.

In some embodiments, the above cell culture medium is for use in amethod as disclosed herein. In some embodiments, the above cell culturemedium is used in a method as disclosed herein as a base media. In someembodiments, the above cell culture medium is used a method as disclosedherein as a feed media.

Expression of Proteins

As noted above, in many instances the cells will be selected orengineered to produce high levels of desired products (e.g., recombinantprotein or antibody). Often, cells will be manipulated by the hand ofman to produce high levels of recombinant protein, for example byintroduction of a gene encoding the protein of interest and/or byintroduction of genetic control elements that regulate expression ofthat gene (whether endogenous or introduced).

Certain proteins may have detrimental effects on cell growth, cellviability or some other characteristic of the cells that ultimatelylimits production of the protein of interest in some way. Even amongst apopulation of cells of one particular type engineered to express aspecific protein, variability within the cellular population exists suchthat certain individual cells will grow better, produce more protein ofinterest, or produce a protein with higher activity levels (e.g.,enzymatic activity). In certain embodiments, a cell line is empiricallyselected by the practitioner for robust growth under the particularconditions chosen for culturing the cells. In some embodiments,individual cells engineered to express a particular protein are chosenfor large-scale production based on cell growth, final cell density,percent cell viability, titer of the expressed protein or anycombination of these or any other conditions deemed important by thepractitioner.

Any protein that is expressible in a host cell may be produced inaccordance with the present teachings. The term “host cell” as usedherein refers to a cell that is manipulated according to the presentinvention to produce a protein of interest as described herein. Aprotein may be expressed from a gene that is endogenous to the cell, orfrom a heterologous gene that is introduced into the cell. A protein maybe one that occurs in nature, or may alternatively have a sequence thatwas engineered or selected by the hand of man.

Proteins that may desirably be expressed in accordance with the presentinvention will often be selected on the basis of an interesting oruseful biological or chemical activity. For example, the presentinvention may be employed to express any pharmaceutically orcommercially relevant enzyme, receptor, antibody, hormone, regulatoryfactor, antigen, binding agent, etc. In some embodiments, the proteinexpressed by cells in culture are selected from antibodies, or fragmentsthereof, nanobodies, single domain antibodies, glycoproteins,therapeutic proteins, growth factors, clotting factors, cytokines,fusion proteins, pharmaceutical drug substances, vaccines, enzymes, orSmall Modular ImmunoPharmaceuticals™ (SMIPs). One of ordinary skill inthe art will understand that any protein may be expressed in accordancewith the present invention and will be able to select the particularprotein to be produced based on his or her particular needs.

Antibodies

Given the large number of antibodies currently in use or underinvestigation as pharmaceutical or other commercial agents, productionof antibodies is of particular interest in accordance with the presentinvention. Antibodies are proteins that have the ability to specificallybind a particular antigen. Any antibody that can be expressed in a hostcell may be produced in accordance with the present invention. In someembodiments, the antibody to be expressed is a monoclonal antibody.

In some embodiments, the monoclonal antibody is a chimeric antibody. Achimeric antibody contains amino acid fragments that are derived frommore than one organism. Chimeric antibody molecules can include, forexample, an antigen binding domain from an antibody of a mouse, rat, orother species, with human constant regions. A variety of approaches formaking chimeric antibodies have been described. See e.g., Morrison etal., Proc. Natl. Acad. Sci. U.S.A. 81, 6851 (1985); Takeda et al.,Nature 314, 452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss etal., U.S. Pat. No. 4,816,397; Tanaguchi et al., European PatentPublication EP171496; European Patent Publication 0173494, UnitedKingdom Patent GB 2177096B.

In some embodiments, the monoclonal antibody is a human antibodyderived, e.g., through the use of ribosome-display or phage-displaylibraries (see, e.g., Winter et al., U.S. Pat. No. 6,291,159 andKawasaki, U.S. Pat. No. 5,658,754) or the use of xenographic species inwhich the native antibody genes are inactivated and functionallyreplaced with human antibody genes, while leaving intact the othercomponents of the native immune system (see, e.g., Kucherlapati et al.,U.S. Pat. No. 6,657,103).

In some embodiments, the monoclonal antibody is a humanized antibody. Ahumanized antibody is a chimeric antibody wherein the large majority ofthe amino acid residues are derived from human antibodies, thusminimizing any potential immune reaction when delivered to a humansubject. In humanized antibodies, amino acid residues in thecomplementarity determining regions are replaced, at least in part, withresidues from a non-human species that confer a desired antigenspecificity or affinity. Such altered immunoglobulin molecules can bemade by any of several techniques known in the art, (e.g., Teng et al.,Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al.,Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92,3-16 (1982)), and are preferably made according to the teachings of PCTPublication WO92/06193 or EP 0239400, all of which are incorporatedherein by reference). Humanized antibodies can be commercially producedby, for example, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex,Great Britain. For further reference, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992), all of which areincorporated herein by reference.

In some embodiments, the monoclonal, chimeric, or humanized antibodiesdescribed above may contain amino acid residues that do not naturallyoccur in any antibody in any species in nature. These foreign residuescan be utilized, for example, to confer novel or modified specificity,affinity or effector function on the monoclonal, chimeric or humanizedantibody. In some embodiments, the antibodies described above may beconjugated to drugs for systemic pharmacotherapy, such as toxins,low-molecular-weight cytotoxic drugs, biological response modifiers, andradionuclides (see e.g., Kunz et al., Calicheamicin derivative-carrierconiuqates, US20040082764 A1).

In general, practitioners of the present invention will select a proteinof interest, and will know its precise amino acid sequence. Any givenprotein that is to be expressed in accordance with the present inventionmay have its own particular characteristics and may influence the celldensity or viability of the cultured cells, may be expressed at lowerlevels than another protein grown under identical culture conditions,and may have different biological activity depending on the exactculture conditions and steps performed. One of ordinary skill in the artwill be able to appropriately modify the steps and compositions used toproduce a particular protein according to the teachings of the presentinvention in order to optimize cell growth and the production and/oractivity level of any given expressed protein.

Introduction of Genes for the Expression of Proteins into Host Cells

Generally, a nucleic acid molecule introduced into the cell encodes theprotein desired to be expressed according to the present invention.Alternatively, a nucleic acid molecule may encode a gene product thatinduces the expression of the desired protein by the cell. For example,introduced genetic material may encode a transcription factor thatactivates transcription of an endogenous or heterologous protein.Alternatively or additionally, an introduced nucleic acid molecule mayincrease the translation or stability of a protein expressed by thecell.

Methods suitable for introducing nucleic acids sufficient to achieveexpression of a protein of interest into mammalian host cells are knownin the art. See, for example, Gething et al., Nature, 293:620-625, 1981;Mantei et al., Nature, 281:40-46, 1979; Levinson et al. EP 117,060; andEP 117,058, each of which is incorporated herein by reference. Formammalian cells, common methods of introducing genetic material intomammalian cells include the calcium phosphate precipitation method ofGraham and van der Erb (Virology, 52:456-457, 1978) or theLipofectamine™ (Gibco BRL) Method of Hawley-Nelson (Focus 15:73, 1993).General aspects of mammalian cell host system transformations have beendescribed by Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983. Forvarious techniques for introducing genetic material into mammaliancells, see Keown et al., Methods in Enzymology, 1989, Keown et al.,Methods in Enzymology, 185:527-537, 1990, and Mansour et al., Nature,336:348-352, 1988.

In some embodiments, a nucleic acid to be introduced is in the form of anaked nucleic acid molecule. For example, the nucleic acid moleculeintroduced into a cell may consist only of the nucleic acid encoding theprotein and the necessary genetic control elements. Alternatively, anucleic acid encoding the protein (including the necessary regulatoryelements) may be contained within a plasmid vector. Non-limitingrepresentative examples of suitable vectors for expression of proteinsin mammalian cells include pCDNA1; pCD, see Okayama, et al. Mol. CellBiol. 5:1136-1142, 1985; pMCIneo Poly-A, see Thomas, et al. Cell51:503-512, 1987; a baculovirus vector such as pAC 373 or pAC 610; CDM8,see Seed, B. Nature 329:840, 1987; and pMT2PC, see Kaufman, et al. EMBOJ. 6:187-195, 1987, each of which is incorporated herein by reference inits entirety. In some embodiments, a nucleic acid molecule to beintroduced into a cell is contained within a viral vector. For example,a nucleic acid encoding the protein may be inserted into the viralgenome (or a partial viral genome). Regulatory elements directing theexpression of the protein may be included with the nucleic acid insertedinto the viral genome (i.e., linked to the gene inserted into the viralgenome) or can be provided by the viral genome itself.

Naked DNA can be introduced into cells by forming a precipitatecontaining the DNA and calcium phosphate. Alternatively, naked DNA canalso be introduced into cells by forming a mixture of the DNA andDEAE-dextran and incubating the mixture with the cells or by incubatingthe cells and the DNA together in an appropriate buffer and subjectingthe cells to a high-voltage electric pulse (e.g., by electroporation). Afurther method for introducing naked DNA cells is by mixing the DNA witha liposome suspension containing cationic lipids. The DNA/liposomecomplex is then incubated with cells. Naked DNA can also be directlyinjected into cells by, for example, microinjection.

Alternatively, naked DNA can also be introduced into cells by complexingthe DNA to a cation, such as polylysine, which is coupled to a ligandfor a cell-surface receptor (see for example Wu, G. and Wu, C. H. J.Biol. Chem. 263:14621, 1988; Wilson et al. J. Biol. Chem. 267:963-967,1992; and U.S. Pat. No. 5,166,320, each of which is hereby incorporatedby reference in its entirety). Binding of the DNA-ligand complex to thereceptor facilitates uptake of the DNA by receptor-mediated endocytosis.

Use of viral vectors containing particular nucleic acid sequences, e.g.,a cDNA encoding a protein, is a common approach for introducing nucleicacid sequences into a cell. Infection of cells with a viral vector hasthe advantage that a large proportion of cells receive the nucleic acid,which can obviate the need for selection of cells which have receivedthe nucleic acid. Additionally, molecules encoded within the viralvector, e.g., by a cDNA contained in the viral vector, are generallyexpressed efficiently in cells that have taken up viral vector nucleicacid.

Defective retroviruses are well characterized for use in gene transferfor gene therapy purposes (for a review see Miller, A. D. Blood 76:271,1990). A recombinant retrovirus can be constructed having a nucleic acidencoding a protein of interest inserted into the retroviral genome.Additionally, portions of the retroviral genome can be removed to renderthe retrovirus replication defective. Such a replication defectiveretrovirus is then packaged into virions which can be used to infect atarget cell through the use of a helper virus by standard techniques.

The genome of an adenovirus can be manipulated such that it encodes andexpresses a protein of interest but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. See, forexample, Berkner et al. BioTechniques 6:616, 1988; Rosenfeld et al.Science 252:431-434, 1991; and Rosenfeld et al. Cell 68:143-155, 1992.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) areknown to those skilled in the art. Recombinant adenoviruses areadvantageous in that they do not require dividing cells to be effectivegene delivery vehicles and can be used to infect a wide variety of celltypes, including airway epithelium (Rosenfeld et al., 1992, citedsupra), endothelial cells (Lemarchand et al., Proc. Natl. Acad. Sci. USA89:6482-6486, 1992), hepatocytes (Herz and Gerard, Proc. Natl. Acad.Sci. USA 90:2812-2816, 1993) and muscle cells (Quantin et al., Proc.Natl. Acad. Sci. USA 89:2581-2584, 1992). Additionally, introducedadenoviral DNA (and foreign DNA contained therein) is not integratedinto the genome of a host cell but remains episomal, thereby avoidingpotential problems that can occur as a result of insertional mutagenesisin situations where introduced DNA becomes integrated into the hostgenome (e.g., retroviral DNA). Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmandand Graham, J. Virol. 57:267, 1986). Most replication-defectiveadenoviral vectors currently in use are deleted for all or parts of theviral E1 and E3 genes but retain as much as 80% of the adenoviralgenetic material.

Adeno-associated virus (AAV) is a naturally occurring defective virusthat requires another virus, such as an adenovirus or a herpes virus, asa helper virus for efficient replication and a productive life cycle.(For a review see Muzyczka et al. Curr. Topics in Micro. and Immunol.,158:97-129, 1992). It is also one of the few viruses that may integrateits DNA into non-dividing cells, and exhibits a high frequency of stableintegration (see for example Flotte et al., Am. J. Respir. Cell. Mol.Biol. 7:349-356, 1992; Samulski et al., J. Virol. 63:3822-3828, 1989;and McLaughlin et al., J. Virol. 62:1963-1973, 1989). Vectors containingas little as 300 base pairs of AAV can be packaged and can integrate.Space for exogenous DNA is limited to about 4.5 kb. An AAV vector suchas that described in Tratschin et al. (Mol. Cell. Biol. 5:3251-3260,1985) can be used to introduce DNA into cells. A variety of nucleicacids have been introduced into different cell types using AAV vectors(see for example Hermonat et al., Proc. Natl. Acad. Sci. USA81:6466-6470, 1984; Tratschin et al., Mol. Cell. Biol. 4:2072-2081,1985; Wondisford et al., Mol. Endocrinol. 2:32-39, 1988; Tratschin etal., J. Virol. 51:611-619, 1984; and Flotte et al., J. Biol. Chem.268:3781-3790, 1993).

When the method used to introduce nucleic acid molecules into apopulation of cells results in modification of a large proportion of thecells and efficient expression of the protein by the cells, the modifiedpopulation of cells may be used without further isolation or subcloningof individual cells within the population. That is, there may besufficient production of the protein by the population of cells suchthat no further cell isolation is needed and the population can beimmediately be used to seed a cell culture for the production of theprotein. Alternatively, it may be desirable to isolate and expand ahomogenous population of cells from a few cells or a single cell thatefficiently produce(s) the protein.

Alternative to introducing a nucleic acid molecule into a cell thatencodes a protein of interest, the introduced nucleic acid may encodeanother polypeptide or protein that induces or increases the level ofexpression of the protein produced endogenously by a cell. For example,a cell may be capable of expressing a particular protein but may fail todo so without additional treatment of the cell. Similarly, the cell mayexpress insufficient amounts of the protein for the desired purpose.Thus, an agent that stimulates expression of the protein of interest canbe used to induce or increase expression of that protein by the cell.For example, the introduced nucleic acid molecule may encode atranscription factor that activates or upregulates transcription of theprotein of interest. Expression of such a transcription factor in turnleads to expression, or more robust expression of the protein ofinterest.

In certain embodiments, a nucleic acid that directs expression of theprotein is stably introduced into the host cell. In certain embodiments,a nucleic acid that directs expression of the protein is transientlyintroduced into the host cell. One of ordinary skill in the art will beable to choose whether to stably or transiently introduce a nucleic acidinto the cell based on his or her experimental needs.

A gene encoding a protein of interest may optionally be linked to one ormore regulatory genetic control elements. In certain embodiments, agenetic control element directs constitutive expression of the protein.In certain embodiments, a genetic control element that providesinducible expression of a gene encoding the protein of interest can beused. The use of an inducible genetic control element (e.g., aninducible promoter) allows for modulation of the production of theprotein in the cell. Non-limiting examples of potentially usefulinducible genetic control elements for use in eukaryotic cells includehormone-regulated elements (e.g., see Mader, S. and White, J. H., Proc.Natl. Acad. Sci. USA 90:5603-5607, 1993), synthetic ligand-regulatedelements (see, e.g. Spencer, D. M. et al., Science 262:1019-1024, 1993)and ionizing radiation-regulated elements (e.g., see Manome, Y. et al.,Biochemistry 32:10607-10613, 1993; Datta, R. et al., Proc. Natl. Acad.Sci. USA 89:10149-10153, 1992). Additional cell-specific or otherregulatory systems known in the art may be used in accordance with theinvention.

One of ordinary skill in the art will be able to choose and, optionally,to appropriately modify the method of introducing genes that cause thecell to express the protein of interest in accordance with the teachingsof the present invention.

Isolation of the Expressed Protein

In general, it will typically be desirable to isolate and/or purifyproteins expressed according to the present invention. In certainembodiments, the expressed protein is secreted into the medium and thuscells and other solids may be removed, as by centrifugation or filteringfor example, as a first step in the purification process.

Alternatively, the expressed protein may be bound to the surface of thehost cell. For example, the media may be removed and the host cellsexpressing the protein are lysed as a first step in the purificationprocess. Lysis of mammalian host cells can be achieved by any number ofmeans well known to those of ordinary skill in the art, includingphysical disruption by glass beads and exposure to high pH conditions.

The expressed protein may be isolated and purified by standard methodsincluding, but not limited to, chromatography (e.g., ion exchange,affinity, size exclusion, and hydroxyapatite chromatography), gelfiltration, centrifugation, or differential solubility, ethanolprecipitation and/or by any other available technique for thepurification of proteins (See, e.g., Scopes, Protein PurificationPrinciples and Practice 2nd Edition, Springer-Verlag, New York, 1987;Higgins, S. J. and Hames, B. D. (eds.), Protein Expression: A PracticalApproach, Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. I.,Abelson, J. N. (eds.), Guide to Protein Purification: Methods inEnzymology (Methods in Enzymology Series, Vol. 182), Academic Press,1997, each of which is incorporated herein by reference). Forimmunoaffinity chromatography in particular, the protein may be isolatedby binding it to an affinity column comprising antibodies that wereraised against that protein and were affixed to a stationary support.Alternatively, affinity tags such as an influenza coat sequence,poly-histidine, or glutathione-S-transferase can be attached to theprotein by standard recombinant techniques to allow for easypurification by passage over the appropriate affinity column. Proteaseinhibitors such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin,pepstatin or aprotinin may be added at any or all stages in order toreduce or eliminate degradation of the protein during the purificationprocess. Protease inhibitors are particularly advantageous when cellsmust be lysed in order to isolate and purify the expressed protein.

One of ordinary skill in the art will appreciate that the exactpurification technique will vary depending on the character of theprotein to be purified, the character of the cells from which theprotein is expressed, and/or the composition of the medium in which thecells were grown.

Pharmaceutical Formulations

In certain preferred embodiments of the invention, produced polypeptidesor proteins will have pharmacologic activity and will be useful in thepreparation of pharmaceuticals. Inventive compositions as describedabove may be administered to a subject or may first be formulated fordelivery by any available route including, but not limited to parenteral(e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial,ophthalmic, transdermal (topical), transmucosal, rectal, and vaginalroutes. Inventive pharmaceutical compositions typically include apurified polypeptide or protein expressed from a mammalian cell line, adelivery agent (i.e., a cationic polymer, peptide molecular transporter,surfactant, etc., as described above) in combination with apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline(PBS). In all cases, the composition should be sterile and should befluid to the extent that easy syringability exists. Preferredpharmaceutical formulations are stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. In general, therelevant carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thepurified polypeptide or protein in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the purified polypeptide or protein expressedfrom a mammalian cell line into a sterile vehicle which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, thepurified polypeptide or protein can be incorporated with excipients andused in the form of tablets, troches, or capsules, e.g., gelatincapsules. Oral compositions can also be prepared using a fluid carrierfor use as a mouthwash. Pharmaceutically compatible binding agents,and/or adjuvant materials can be included as part of the composition.The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. Formulations fororal delivery may advantageously incorporate agents to improve stabilitywithin the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the inventive compositions comprisinga purified polypeptide or protein expressed from a mammalian cell lineand a delivery agent are preferably delivered in the form of an aerosolspray from a pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer. Thepresent invention particularly contemplates delivery of the compositionsusing a nasal spray, inhaler, or other direct delivery to the upperand/or lower airway. Intranasal administration of DNA vaccines directedagainst influenza viruses has been shown to induce CD8 T cell responses,indicating that at least some cells in the respiratory tract can take upDNA when delivered by this route, and the delivery agents of theinvention will enhance cellular uptake. According to certain embodimentsof the invention the compositions comprising a purified polypeptideexpressed from a mammalian cell line and a delivery agent are formulatedas large porous particles for aerosol administration.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the purified polypeptide or protein anddelivery agents are formulated into ointments, salves, gels, or creamsas generally known in the art.

The compositions can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

In some embodiments, the compositions are prepared with carriers thatwill protect the polypeptide or protein against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active polypeptide or proteincalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier.

The polypeptide or protein expressed according to the present inventioncan be administered at various intervals and over different periods oftime as required, e.g., one time per week for between about 1 to 10weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or6 weeks, etc. The skilled artisan will appreciate that certain factorscan influence the dosage and timing required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Generally, treatment of a subjectwith a polypeptide or protein as described herein can include a singletreatment or, in many cases, can include a series of treatments. It isfurthermore understood that appropriate doses may depend upon thepotency of the polypeptide or protein and may optionally be tailored tothe particular recipient, for example, through administration ofincreasing doses until a preselected desired response is achieved. It isunderstood that the specific dose level for any particular animalsubject may depend upon a variety of factors including the activity ofthe specific polypeptide or protein employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

The present invention includes the use of compositions for treatment ofnonhuman animals. Accordingly, doses and methods of administration maybe selected in accordance with known principles of veterinarypharmacology and medicine. Guidance may be found, for example, in Adams,R. (ed.), Veterinary Pharmacology and Therapeutics, 8^(th) edition, IowaState University Press; ISBN: 0813817439; 2001.

Pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

EXAMPLES Example 1: Identification of the Metabolic Byproducts,Accumulating in Fed-Batch Cultures, which have Inhibitory Effects onGrowth of Mammalian Cells in Culture Goal:

This experiment was carried out to identify the major growth inhibitors(metabolic byproducts) accumulating in the glucose restricted andconventional fed-batch cultures of mammalian cells, using globalmetabolite profiling approaches.

Materials and Methods: Cells and Medium

CHO cells comprising a glutamine synthase expression system(commercially available from Lonza) (hereafter GS-CHO, Cell line A) andexpressing a recombinant antibody were used in the current experiment.Two types of medium were used in this experiment. First medium is“Medium A” which is used for inoculation of the experiment on day 0 ofthe culture. Second medium is “Medium B” which is the enriched nutrientmedia used as a feed medium for conventional and HIPDOG fed-batchprocesses (described in the next section).

Medium A is a fortified version of insulin-free Medium 9 (U.S. Pat. No.7,294,484, table 14), with slight differences in concentrations ofsodium bicarbonate and potassium chloride, and containing Pluronic F68instead of polyvinyl alcohol. It was fortified by adding 10%glutamine-free Medium 5 (U.S. Pat. No. 7,294,484, table 7), and byfurther raising the concentrations of eight amino acids (Glu, Tyr, Gly,Phe, Pro, Thr, Trp and Val). The concentrations of amino acids arelisted in the Table 1 below.

TABLE 1 Concentration of Amino Acids in Medium A Amino AcidsConcentration in Medium A (mM) alanine 0.4 arginine 5.3 asparagine•H2O21.1 aspartic acid 2.3 cysteine•HCl•H2O 0.4 cystine•2HCl 1.5 glutamicacid 0 monosodium glutamate 2.0 glutamine 0 glycine 3.6histidine•HCl•H2O 2.7 isoleucine 5.4 leucine 9.4 lysine•HCl 8.9methionine 3.1 phenylalanine 4.5 proline 9.1 serine 11.8 threonine 10.8tryptophan 2.3 tyrosine•2Na•2H2O 5.1 valine 10.3

Medium B has the same composition as Medium 5 (U.S. Pat. No. 7,294,484,table 7), but with higher levels of the amino acids (by a factor of2.5). The concentrations of amino acids in Medium B are shown in Table2.

TABLE 2 Concentration of Amino Acids in Medium B Amino AcidsConcnetration in Medium B (mM) alanine 6.0 arginine 32.9 asparagine•H₂O54.0 aspartic acid 15.0 cysteine•HCl•H₂O 0.0 cystine•2HCl 4.7 glutamicacid 6.0 monosodium glutamate 0.0 glutamine 0.0 glycine 6.0histidine•HCl•H₂O 10.5 isoleucine 27.0 leucine 38.9 lysine•HCl 30.0methionine 12.0 phenylalanine 15.0 proline 18.0 serine 45.2 threonine24.0 tryptophan 4.8 tyrosine•2Na•2H₂O 12.0 valine 24.0

Bioreactor Setup

Two conditions were employed including conventional fed-batch processand a glucose restricted fed-batch process. In the glucose restrictedfed batch process (hereafter HIPDOG culture), glucose was limited byusing the HIPDOG technology (Gagnon et al, 2011). The pH dead-band usedwhile the HIPDOG control was operational was 7.025+1-0.025.

The conventional process was identical to the HIPDOG process withrespect to inoculum cell density targeted (1E6 cells/mL), the mediaused, culture volume (14 the amount of feed added daily to the culture,and the process parameters including the temperature (36.5 C), pH(6.9-7.2) and the agitation rate (267 rpm). The two cultures onlydiffered in their glucose levels. In the conventional culture, glucosewas maintained at greater than 2 g/L between days 2 through 5, while inthe HIPDOG culture glucose was consumed by the cells naturally until theglucose level fell to a point at which the cells began to also consumelactic acid (observed by a slight rise in pH of the culture) and theHIPDOG technology/feeding strategy commenced. Post day 5 the glucoselevels in both the conditions were maintained at concentrations above 2g/L by feeding glucose as necessary and were treated similarly until day12. Viable cell density, lactate and ammonia concentration in the cellculture medium were measured on a daily basis for both the conditions.The base medium used is Medium A and the feed medium used was Medium B.

For metabolomic analysis, spent medium samples and the cell pelletsamples were collected and analyzed from duplicate reactors runs,performed for each condition. Time points considered for the analysisinclude days 0, 2, 3, 5, 7, 9 and 10. Metabolomic approach used employedboth NMR (groups 4 and 5 of Table 3), LC/MS and GC/MS (groups 1 to 3 oftable 3) techniques to assess the relative levels of metabolites atdifferent time points of the culture. The details of the samplepreparation and the type of equipment/methods used for NMR, LC/MS andGC/MS analysis are described below. The relative levels (fold changes)of all metabolites were measured and calculated. The relative levelswere determined in both the spent medium and cell pellet samples, whichwere calculated based on fold changes compared to the level of themetabolite when first detected. The fold changes were used to identifythe metabolites that were accumulating to very high levels by day 7 ofthe HIPDOG and the conventional fed-batch culture.

Methods for Metabolomic Analysis

Liquid/Gas Chromatography with Mass Spectrometry

Sample preparation was conducted using a methanol extraction to removethe protein fraction while allowing maximum recovery of small molecules.The resulting extract was dried under vacuum and subsequently used forsample preparation for the appropriate instrument, either LC/MS orGC/MS.

The LC/MS portion of the platform was based on a Waters ACQUITY UPLC anda Thermo-Finnigan LTQ mass spectrometer, which consisted of anelectrospray ionization (ESI) source and linear ion-trap (LIT) massanalyzer. The sample was analyzed independently in both positive andnegative ion modes. Sample was reconstituted in acidic conditions forpositive ion mode and was gradient eluted using water and methanol, bothcontaining 0.1% Formic acid, whereas for negative ion mode sample wasreconstituted in basic extracts, which also used water/methanol,contained 6.5 mM ammonium bicarbonate for gradient elution. The MSanalysis alternated between MS and data-dependent MS² scans usingdynamic exclusion.

The samples destined for GC/MS analysis were re-dried under vacuumdesiccation for a minimum of 24 hours prior to being derivatized underdried nitrogen using bistrimethyl-silyl-triflouroacetamide (BSTFA). TheGC column was 5% phenyl and the temperature ramp is from 40° to 300° C.in a 16 minute period. Samples were analyzed on a Thermo-Finnigan TraceDSQ fast-scanning single-quadrupole mass spectrometer using electronimpact ionization. The instrument was tuned and calibrated for massresolution and mass accuracy on a frequent basis.

The data was extracted from the raw mass spec data files and peaks wereidentified. Subsequently, the peaks were annotated and quantified(arbitrary intensity values) with compound information by comparison tolibrary entries of purified standards or recurrent unknown entities. Thecombination of chromatographic properties and mass spectra gave anindication of a match to the specific compound or an isobaric entity.

NMR Sample Preparation, Data Acquisition and Processing

1000 μL of each sample was filtered using Nanosep 3K Omegamicrocentrifuge filter tubes for 60 minutes, and 630 μL of the filteredsample was used for NMR analysis. These filters are preserved withglycerol, and as such some trace amounts of glycerol may appear in theanalysis. Internal standard solution was added to each sample solution,and the resulting mixture was vortexed for 30 s. 700 μL of thecentrifuged solution was transferred to an NMR tube for dataacquisition.

NMR spectra were acquired on a Varian four-channel VNMRS 700 MHz NMRspectrometer equipped with a cryogenically cooled 1H/13C tripleresonance biomolecular probe with auto tuning. The pulse sequence usedwas a 1 D-tnnoesy with a 990 ms presaturation on water and a 4 sacquisition time. Spectra were collected with 32 transients and 4steady-state scans at 298 K.

Spectra were processed and .cnx files were generated using the Processormodule in Chenomx NMR Suite 8.0. Compounds were identified andquantified using the Profiler module in Chenomx NMR Suite 8.0 with theChenomx Compound Library version 9, containing 332 compounds. Forreporting purposes, the profiled concentrations have been corrected toreflect the composition of the original sample, instead of the contentsof the NMR tube. During sample preparation, each sample is diluted byintroducing an internal standard and, where necessary, to increase theanalyzed volume of a small sample.

Results:

Initially cells grew exponentially in both conventional and HIPDOGcultures and attained peak cells densities on day 6 and day 7,respectively, with HIPDOG culture peaking at much higher cell densities(FIG. 1). The lactate levels in the HIPDOG process remained low due toapplication of the HIPDOG control (between day 2-day 5) whereas thelactate levels accumulated to very high levels in case of theconventional fed-batch culture. Ammonia was also maintained at lowlevels during the conventional and HIPDOG culture by the use of cellscomprising a glutamine synthetase expression system. The titer (amountof protein of interest per liter of cell culture medium) was measured atthe end of the culture (Day 12). The HIPDOG culture attained highertiter compared to the conventional process. The differences in the celldensities and titer values are likely an outcome of the differences inthe lactate accumulations observed between the two cultures.

The metabolites that were accumulating to high levels on day 7 of theHIPDOG culture were identified based on the fold changes measured usingthe global metabolite profiling techniques. For each of the aboveidentified metabolites, the concentration at which the metaboliteaffects the growth of the cell negatively was determined throughspike-in experiments using purified compounds (see example 3). Theresults of these experiments were used to narrow the list of theputative novel inhibitors. A total of 9 inhibitors were identified bythis process. The list of the 9 metabolites identified as potentialinhibitors as well as their potential metabolic source in the cellculture medium are reported in Table 3.

Table 3 Shows the Names and the Functional Classes of the NineMetabolites Identified as Putative Growth Inhibitors Accumulating in theGS-CHO Fed-Batch Cultures

Group Metabolite Functional Class 1 3-(4-hydroxyphenyl)lactatePhenylalanine & tyrosine (HPLA) metabolism 4-hydroxyphenylpyruvatePhenyllactate (PLA) Phenylalanine metabolism 2 Indolelactate(indole-3-lactate) Tryptophan metabolism Indolecarboxylic acid(indole-3- carboxylic acid) 3 Homocysteine Methionine metabolism2-hydroxybutyric acid 4 Isovalerate Leucine 5 Formate Serine, Threonineand Glycine

Example 2: Determination of the Concentration to which the PutativeInhibitors Accumulate in Late Stages of HIPDOG Fed-Batch Cultures Goal:

This experiment was carried out to assess the concentrations of newlyidentified putative growth inhibitors (metabolic byproducts) atdifferent time points in the HIPDOG fed-batch cultures of GS-CHO cells.

Materials and Methods:

Experimental setup is same as the one defined in Example 1.Quantification was performed by LC/MS and GC/MS methods for metabolitesin the first two groups of Table 1 and NMR technology was used forquantification of the metabolites in groups 4 and 5. For first twogroups of the metabolites listed in the metabolite column of Table 1,purified compounds were obtained commercially and solutions of thesecompounds at known concentrations were prepared using the Medium A asthe base solvent. Using a similar LC/MS and GC/MS global metaboliteprofiling approach as that used for inhibitor identification andrelative quantification (see Example 1), independent calibration curvesfor four metabolites were prepared. These calibration curves aremathematical correlations of the actual amounts of the metabolite usedin LC/MS and GS/MS techniques to the intensity values generated by thesame. The correlations are subsequently used along with the intensityvalues generated in Example 1 to calculate the concentration of themetabolites at different time points in the culture.

Results:

The concentrations of newly identified metabolites from the first twogroups of Table 3 were determined using the calibration curvesdeveloped, and the concentrations for the metabolites listed in groups 4and 5 were determined by NMR technology as discussed in the Materialsand Methods section of Example 1. The concentration of six putativeinhibitors (phenyllactate, 3-(4-hydroxyphenyl)lactate,4-hydroxyphenylpyruvate, indolelactate, isovalerate and formate) on day7 of the HIPDOG fed-batch cultures are listed Table 4.

TABLE 4 concentrations of six metabolites on day 7 of the HIPDOG cultureDay 7 Concentration Metabolite (mM) 3-(4hydroxyphenyl)lactate (HPLA)0.38 4-hydroxyphenylpyruvate 0.08 Phenyllactate (PLA) 0.20 Indolelactate0.26 Isovalerate 2.41 Formate 3.97

Example 3: Experiment to Establish the Growth Suppressive Effect ofIdentified Putative Inhibitors at the Concentrations Detected on Day 7of the HIPDOG Fed-Batch Culture Goal:

This experiment was carried out to assess the effect of the newlyidentified putative inhibitors, at the concentrations determined on day7 of the HIPDOG culture, on growth of GS-CHO cells in culture. Theindependent effect of the nine metabolites (phenyllactate,3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, indolelactate,indolecarboxylic acid, homocysteine, 2-hydroxybutyric acid, isovalerateand formate) on growth of cells was tested first. Subsequently,synergistic effect for four metabolites (phenyllactate,3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate and indolelactate)on cell growth was tested.

Materials and Methods:

GS-CHO cells producing a recombinant antibody were inoculated at lowviable cell densities (0.1E6 cells/mL) in various conditions in 5 mlvolume, 6-well plate cultures. These conditions include fresh Medium Aor fresh Medium A spiked-in with four putative inhibitors atconcentrations detected on day 7 of the HIPDOG culture of Example 1(phenyllactate at 0.2 mM, 3-(4-hydroxyphenyl)lactate at 0.38 mM,4-hydroxyphenylpyruvate at 0.08 mM and indolelactate at 0.26 mM). In aseparate experiment, GS-CHO cells producing a recombinant antibody wereinoculated at low viable cell densities in fresh Medium A spiked-in withdifferent concentrations of 9 inhibitors (indolecarboxylic acid,homocysteine, 2-hydroxybutyric acid, phenyllactate,3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, indolelactate,isovalerate and formate) with one inhibitor at a time per condition. ThepH of the Medium A, spiked-in with any one of the nine metabolites orany combination of the nine metabolites, was adjusted to 7 beforeinoculating the cells (on day 0). The concentrations tested are:

-   -   indolecarboxylic acid: 0, 0.5 and 1 mM    -   homocysteine: 0, 0.5 and 1 mM    -   2-hydroxybutyric acid: 0, 1 and 5 mM    -   phenyllactate: 0, 1 and 5 mM    -   3-(4-hydroxyphenyl)lactate: 0, 0.1, 0.3, 0.5, 1 and 5 mM    -   4-hydroxyphenylpyruvate: 0, 0.05, 0.1 and 0.25 mM    -   indolelactate: 0, 1 and 3 mM    -   isovalerate: 0, 1, 2.5 and 5 mM    -   formate: 0, 2, 4 and 6 mM

For indolelactate, the stock solution (500 mM) was prepared in DMSO.Hence, pure DMSO spike-in conditions (‘DMSO cont’ or DMSO control) wereincluded for every concentration of indolelactate tested, so as tocontrol for the effect of DMSO on the growth of the cells. All theconditions were run in duplicates or triplicates. Growth of the cells inabove described conditions was monitored for 5 or 6 days.

Results:

The independent effect of the all the nine inhibitors on growth of theGS-CHO cells was investigated (FIG. 2, FIG. 3, FIG. 4 and FIG. 5).Indolecarboxylic acid and 4-hydroxyphenylpyruvate were observed to havea potent negative effect on growth of cells at concentrations of 1 mM orlower. Modest inhibition of growth was observed when GS-CHO cells wereexposed to either homocysteine, 2-hydroxybutyric acid or3-(4-hydroxyphenyl)lactate at concentrations higher than 0.5 mM or 1 mM.L-phenyllactate had a mild effect on growth of cells at 1 mMconcentration. Indolelactate showed little or no effect on growth ofcells at concentrations of 3 mM or below. Formate had a negative effecton growth at concentrations 2 mM and beyond. Isovalerate had asignificant negative effect on growth of cells at concentrations above 1mM.

Earlier experiment showed that GS-CHO cells when treated independentlywith each of the seven putative inhibitors from groups 1 to 3, theconcentration at which cell growth is inhibited was generally muchhigher than the concentration measured on day 7 of the HIPDOG fed-batchculture. Therefore, the effect of these inhibitors when treated incombination was subsequently evaluated. Interestingly, on treating thecells with the combination of four metabolites (phenyllactate,4-hydroxyphenyllactate, 3-(4-hydroxyphenyl)lactate and indolelactate),at concentrations detected on day 7 of HIPDOG culture, cell growth wassignificantly inhibited when compared to cell growth in the fresh medium(FIG. 6). This data indicates that the above four metabolites act in asynergistic manner to inhibit growth of GS-CHO cells.

Since these four metabolites are by-products of phenylalanine, tyrosineand tryptophan metabolism, reducing the concentrations of theseprecursor amino acids in culture can limit the formation ofcorresponding inhibitory metabolites. Further, since methioninemetabolites (homocysteine and 2-hydroxybutyrate) and leucine, serine,threonine and glycine metabolites (isovalerate and formate) also have anegative effect on cell growth, reducing methionine, leucine, serine,threonine and glycine levels in culture could potentially limit theformation of these metabolites and promote cell growth.

Example 4: Reduction of the Growth Suppressive Effect of the NewlyIdentified Inhibitors by Nutrient Limitation Strategies in Fed-BatchCulture Goal:

This experiment was performed to reduce the formation of newlyidentified inhibitors by limiting the supply of the carbon sourcesresponsible for their biosynthesis. The goal of this experiment was toassess if such reduction in inhibitor formation relieves the growthsuppression in the late stages of the culture, resulting in increasedmaximum viable cell densities in the fed-batch cultures.

Materials and Methods: Cells and Bioreactor Setup

GS-CHO cells expressing a recombinant antibody (cell line A) were usedin the current experiment. Two conditions were tested as part of thisexperiment: A) HIPDOG fed-batch culture with low levels of four aminoacids ((tyrosine, methionine, phenylalanine and tryptophan) (Low AAcondition)), B) HIPDOG fed-batch culture with normal amino acidsconcentrations (control HIPDOG condition). Exponentially growing cellsfrom seed culture were inoculated at 1×10⁶ cells/mL into each productionbioreactor. For both the conditions, HIPDOG strategy was in operationbetween day 2 and day 7 of the culture. In the low amino acid condition,the concentrations of tyrosine, tryptophan, phenylalanine and methioninewere maintained between 0.5 mM and 1 mM for first seven days of theculture after which they were adjusted to the levels of each of thoseamino acids in the control HIPDOG condition. Post day 7 both theconditions were treated similarly. Viable cell density, lactate, ammoniaand amino acid concentrations were measured on a daily basis (aminoacids measured only for first seven days). For both conditions, theinoculum viable cell density targeted (1×10⁶ cells/mL), and the culturevolume (1 L) and the process parameters including the temperature (36.5C), pH (6.9-7.2) and the agitation rate (267 rpm) were identical. Thebase medium used in the control HIPDOG condition was Medium A and thatused in Low AA condition was the modified version of Medium A with lowconcentrations of the four amino acids (tyrosine, tryptophan,phenylalanine and methionine at approximately 0.6 mM). The feed mediumused for control HIPDOG culture was Medium B. For the Low AA condition,either the original Medium B or a modified version of Medium B withhigher concentration of four amino acids (tyrosine, tryptophan,methionine and phenylalanine) was formulated and used as feed media (60%higher for methionine and -100% higher levels for tyrosine, tryptophanand phenylalanine as compared to original feed Medium B). The levels ofthe four amino acids in the modified version of Medium B was configuredbased on the cell specific consumption rates for the four amino acidsand the previously determined feeding schedule for the cell line A in aHIPDOG process. Amino acid concentrations were measured every day usinga UPLC based amino acid method which is described in detail below. Basedon the level of amino acids at a given sampling point and the feedingschedule, one of the two types of medium B (original or higherconcentration) was chosen as the feed medium till next sampling pointsuch that the concentration for the four amino acids are between 0.5mM-1 mM at the next sampling time point.

Amino Acid Analysis

10 μL of either a standard amino acid mix solution or a spent mediumsample (10 times diluted sample) was mixed with 70 μL of AccQ•Tag Ultraborate buffer (Waters UPLC AAA H-Class Applications Kit [176002983]),and 20 μL of AccQ•Tag reagent previously dissolved in 1.0 mL of AccQ•TagUltra reagent diluent was added. The reaction was allowed to proceed for10 min at 55° C. Liquid chromatographic analysis was performed on aWaters Acquity UPLC system, equipped with a binary solvent manager, anautosampler, a column heater and a PDA detector. The separation columnwas a Waters AccQ•Tag Ultra column (2.1 mm i.d.×100 mm, 1.7 μmparticles). The column heater was set at 55° C., and the mobile phaseflow rate was maintained at 0.7 mL/min. Eluent A was 10% AccQ•Tag Ultraconcentrate solvent A, and eluent B was 100% AccQ•Tag Ultra solvent B.The nonlinear separation gradient was 0-0.54 min (99.9% A), 5.74 min(90.0% A), 7.74 min (78.8% A), 8.04-8.64 min (40.4% A), 8.73-10 min(99.9% A). One microliter of sample was injected for analysis. The PDAdetector was set at 260 nm. The previously determined elution times forthe amino acids are used to identify the specific amino acid peaks onthe chromatogram for each sample. The amino acid concentrations wereestimated using the area under the peak and the calibration curvegenerated using the standard solution (Amino Acids Standard H, ThermoScientific, PI-20088).

Results:

The concentrations of the amino acids were successfully maintainedbetween 0.5 mM-1 mM in Low AA conditions (FIG. 8 and FIG. 9). The aminoacid concentrations in the control HIPDOG process remained high over thecourse of the culture. As shown in FIG. 7, the cell densities in the LowAA condition peaked around 35×10⁶ cells/ml on day 7 whereas the celldensities in control HIPDOG condition peaked around 32×10⁶ cells/mL. Theharvest titer levels in the Low AA condition (5.3 g/L) were 18% higherthan the control condition (4.5 g/L). Clearly, limiting the amino acidsupply increased the cell densities (and thereby the titer) in the latestages of the cultures.

Example 5 demonstrates that such an increase in growth and productivitycan be explained as a result of reduced production of the newlyidentified inhibitors.

Example 5: Demonstrating (i) the Reduced Accumulation of the NewlyIdentified Inhibitors Through the Limitation of Amino Acids and (ii) theReproducibility of the Positive Effect of Such Limitation of InhibitoryMetabolites on Growth of GS-CHO Cells (Cell Line A) in Fed-BatchCultures Goal:

The main goal of this example is to demonstrate the reduction in theaccumulation of the newly identified inhibitors in fed-batch cultures bylimiting the supply of the carbon sources (amino acids) responsible fortheir biosynthesis. In this example, two experiments are included todemonstrate that such reduction in the levels of the newly identifiedinhibitors through limitation of four amino acids or eight amino acidsrelieves the growth suppression in the late stages of the fed-batchculture resulting in increased maximum viable cell densities.

Materials and Methods: Cells and Bioreactor Setup

Cell line A was used in the two experiments performed as part of thisexample.

Three conditions were tested in the first experiment:

A) HIPDOG fed-batch culture with low levels of tyrosine, methionine,phenylalanine and tryptophan (Low 4AA+HIPDOG condition),B) HIPDOG fed-batch culture with low levels of tyrosine, methionine,phenylalanine, tryptophan, leucine, serine, threonine and glycine (Low8AA+HIPDOG condition), and,C) two HIPDOG fed-batch cultures with normal amino acids concentrations(HIPDOG 1 and HIPDOG 2).

In a second experiment two conditions were tested:

A) HIPDOG fed-batch culture with low levels of tyrosine, methionine,phenylalanine, tryptophan, leucine, serine, threonine and glycine (Low8AA+HIPDOG condition), and,B) HIPDOG fed-batch culture with low levels of tyrosine, methionine,phenylalanine and tryptophan (Low 4AA+HIPDOG condition).

First experiment was run for 12 days whereas the second experiment wasrun for 16 days. In both the experiments, exponentially growing cellsfrom seed cultures were inoculated at 1×10⁶ cells/mL into eachproduction bioreactor. For all the conditions, HIPDOG control was inoperation between day 2 and day 7 of the culture. In the low amino acidconditions, the concentrations of above mentioned four or eight aminoacids were maintained between 0.5 mM and 1 mM for first seven days ofthe culture after which they were adjusted to the levels closer to therespective amino acids in the control HIPDOG conditions. Post day 7,both the conditions were treated similarly. Viable cell, ammonia andamino acid concentrations were measured on daily basis. For all theconditions, the inoculum viable cell density targeted (1×10⁶ cells/mL),the culture volume (1 L), and the process parameters including thetemperature (36.5 C), pH (6.9-7.2) and agitation rate (259 rpm) wereidentical. The base medium used in the HIPDOG condition was Medium A andthat used in low amino acid conditions was the modified version ofMedium A with low concentrations of either four amino acids (tyrosine,tryptophan, phenylalanine and methionine at approximately 0.6 mM) oreight amino acids (tyrosine, tryptophan, phenylalanine, methionine,leucine, serine, threonine and glycine at approximately 0.6 mM). Thefeed medium used for all the conditions was Medium B. Amino acidconcentrations were measured every day using UPLC based amino acidmethod as described in Example 4. In the low amino acid conditions,based on the level of amino acids at a given sampling point and thefeeding schedule to be followed, concentrated solutions of the aminoacids were supplemented to the conditions such that the concentrationfor the four amino acids or the eight amino acids in corresponding lowamino acid conditions are between 0.5 mM-1 mM at the next sampling timepoint. Spent medium samples from various conditions across both theexperiments were analyzed for the levels of the newly identifiedinhibitors using the NMR technology described in the Materials andMethods section of Example 1.

Results:

In the first experiment, the concentrations of the amino acids weresuccessfully maintained between 0.5 mM-1 mM in both Low 4 AA and Low 8AA conditions until day 7 of the fed-batch cultures (FIGS. 11-14). Suchlimitation of amino acid levels in the two conditions resulted in lowerlevels of accumulation of the newly identified metabolites (FIG. 15 andFIG. 16). Isovalerate, formate, 3-(4-hydroxyphenly)lactate andindole-3-lactate were specifically profiled. Isovalerate and formate arebyproducts of leucine, serine, glycine and threonine, which arecontrolled at low levels only in Low 8 AA condition. These amino acidsare not controlled at low levels in the Low 4 AA condition.Correspondingly, significantly lower concentrations of isovalerate wereonly seen in the Low 8 AA condition. The levels isovalerate were higherin control HIPDOG conditions and the Low 4 AA condition (FIG. 15B).Formate levels were similar across the all conditions on day 7 of theculture; however, on a per cell basis the amount of formate produced islower in Low 8 AA condition compared to HIPDOG conditions. The other twoinhibitors profiled, 3-(4-hydroxyphenly)lactate and indole-3-lactate,are byproducts of the amino acids phenylalanine and tryptophan, whichare controlled at lower levels in both Low 8 AA and Low 4 AA conditions.Significantly lower concentrations of these two inhibitors were observedin both Low 8 AA and Low 4 AA conditions compared the HIDPOG conditionsat day 7 (FIGS. 15A and 16).

The cells in Low 8 AA and Low 4 AA conditions grew better than thecontrol conditions (HIPDOG1 and HIPDOG 2) peaking at cell densities50×10⁶ cells/mL and 45×10⁶ cells/mL, respectively, on day 9 whereas thecell densities in control HIPDOG conditions peaked around 32×10⁶cells/mL (FIG. 10A). Such an increase in the cell growth observed in thelate stages of the low amino acid conditions can be explained as anoutcome of the reduced inhibitor accumulations in the culture (FIGS. 15and 16). In addition, the low amino conditions had higher titer comparedto the control HIPDOG conditions until day 9, which then tapered off tomatch the titer levels of HIPDOG conditions by the end of the culture(FIG. 10B). The post day 9 reduction in the protein production in thelow amino acid conditions was attributed to the near exhaustion oftyrosine levels in the cultures post day 9 (FIG. 11A).

The second experiment was performed to verify and reproduce theincreased positive effect of limiting leucine, serine, glycine andthreonine in Low 8 AA condition when compared to Low 4 AA condition(FIG. 17). Only two conditions were tested in this experiment includingthe Low 8 AA and Low 4 AA condition using the cell line A. The eightamino acids or the four amino acids were controlled between 0.5 mM and 1mM until day 7 of the culture. Amino acid data is not shown for thisexperiment but is similar to the amino acid profiles observed for thetwo conditions in above experiment (FIGS. 11-14) except for tyrosinewhich was not exhausted in this experiment. The cells grew better in theLow 8 AA condition peaking at 42×10⁶ cells/mL on day 9 whereas the celldensities in Low 4 AA condition peaked around 33×10⁶ cells/mL (FIG. 17).Such an increased cell density also translated into higher levels ofharvest titer in the Low 8 AA conditions when compared to Low 4 AAcondition.

Example 6: Demonstrating (i) the Reduction in the Accumulation of theNewly Identified Inhibitors Through the Limitation of Amino Acids and(ii) the Positive Effect of Such Limitation of Inhibitory Metabolites onGrowth of a Different GS-CHO Cell Line (Cell Line B) in Fed-BatchCultures Goal:

This experiment was performed to demonstrate that the growth beneficialeffects of limiting the levels of certain amino acids on the growth ofcells were not specific to one cell line (cell line A) but are moregeneral and can be applied to other cell lines. Two nutrient limitationsexperiments were performed as part of this example using a different CHOcell line (cell line B) producing a different recombinant antibody. Thegoal of these experiments was to show that in fed-batch cultures, thecontrol of amino acids at lower levels results in reduced inhibitoraccumulations and such low accumulations of inhibitors can explain theincreased viable cell densities and protein titers that were seen in thelow amino acid fed-batch cultures.

Materials and Methods: Cells and Bioreactor Setup

A new GS-CHO cell line (cell line B) expressing a different recombinantantibody was used in this example. Two experiments were performed tounderstand the effect of simultaneously limiting either four or eightamino acids. In first experiment, two conditions were tested: A) HIPDOGfed-batch culture with low levels of eight amino acids includingtyrosine, methionine, phenylalanine, tryptophan, leucine, serine,glycine and threonine (Low 8 AA+HIPDOG condition) and B) HIPDOGfed-batch culture with normal amino acids concentrations (HIPDOGcondition). In the second experiment, two conditions were tested: A)HIPDOG fed-batch culture with low levels of four amino acids includingtyrosine, methionine, phenylalanine and tryptophan (Low 4 AA+HIPDOGcondition) and B) HIPDOG fed-batch culture with normal amino acidsconcentrations (HIPDOG condition). First experiment was run for 12 dayswhereas the second experiment was only run till day 8 of the culture.

Exponentially growing cells from seed cultures were inoculated at 1×10⁶cells/mL into each production bioreactor. For all the conditions, HIPDOGstrategy was in operation between day 2 and day 7 of the culture. In thelow amino acid conditions, the concentrations of above mentioned four oreight amino acids were maintained between 0.5 mM and 1 mM for firstseven days of the culture after which they were adjusted to the levelscloser to the respective amino acids in the control HIPDOG condition.Post day 7, both the conditions were treated similarly. Viable cell,ammonia and amino acid concentrations were measured on daily basisthroughout the culture. For all the conditions, the inoculum viable celldensity targeted (1×10⁶ cells/mL), and the culture volume (1 L) and theprocess parameters including the temperature (36.5 C), pH (6.9-7.2) andthe agitation rate (259 rpm) were identical. The base medium used in theHIPDOG condition was Medium A and that used in Low AA conditions was themodified version of Medium A with low concentrations of either fouramino acids (tyrosine, tryptophan, phenylalanine and methionine atapproximately 0.6 mM) or eight amino acids (tyrosine, tryptophan,phenylalanine, methionine, leucine, serine, threonine and glycine atapproximately 0.6 mM). The feed medium used for all the conditions wasMedium B. Amino acid concentrations were measured every day using UPLCbased amino acid method as described in Example 4. In the low amino acidconditions, based on the level of amino acids at a given sampling pointand the feeding schedule to be followed, concentrated solutions of theamino acids were supplemented to the conditions such that theconcentration for the four amino acids or the eight amino acids incorresponding conditions are between 0.5 mM-1 mM at the next samplingtime point. Spent medium samples from various conditions across both theexperiments of this Example were analyzed to quantitate the levels ofthe newly identified inhibitors using the NMR technology, as describedin the Materials and Methods section of Example 1.

Results:

In the first experiment, the concentrations of the eight amino acidswere successfully maintained between 0.5 mM-1 mM in Low 8 AA conditionuntil day 7 of the fed-batch cultures. Amino acid culture profiles arenot shown for this experiment but are similar to those observed for thesimilar conditions in Experiment 1 of Example 5 (FIGS. 11-14) except fortyrosine which was not exhausted in this experiment. Such limitation ofamino acid levels in the Low 8 AA condition resulted in lower levels ofbiosynthesis and reduced accumulation of the newly identifiedmetabolites. Four of the nine inhibitors listed in Table 3 were profiled(FIG. 19 and FIG. 20). In Low 8 AA condition, significantly loweraccumulations of isovalerate, 3-(4-hydroxyphenly)lactate andindole-3-lactate were observed compared to control HIPDOG condition.Formate levels were observed to be higher in Low 8 AA condition on day10 of the culture, however, on a per cell basis, the amount of formateproduced was similar in the Low 8 AA condition compared to the HIPDOGcondition. The cells in Low 8 AA conditions grew better than the controlcondition (HIPDOG) peaking at cell densities of 40×10⁶ cells/mL on day 9whereas the cell densities in control HiPDOG conditions peaked around32×10⁶ cells/mL (FIG. 18A). Further, the increased growth observed inLow 8 AA condition translated into higher titer levels compared to thecontrol HIPDOG condition (FIG. 18B). Such an increase in the cell growthand productivity observed in the low amino acid condition can beexplained as an outcome of the reduced inhibitor biosynthesis andaccumulation (FIGS. 19 and 20).

The second experiment was performed to investigate the effect ofcontrolling the levels of four amino acids (tyrosine, phenylalanine,tryptophan and methionine) between 0.5 mM-1 mM (Low 4 AA+HIPDOG) ongrowth and productivity of cell line B, when compared to control HIPDOGcondition (FIGS. 21-23). Amino acid culture profiles are not shown forthis experiment but are similar to those observed for the similarconditions in Experiment 1 of Example 5 (FIGS. 11-14) except fortyrosine which was not exhausted in this experiment. Such limitation ofamino acid levels in the Low 4 AA condition resulted in lower levels ofbiosynthesis and reduced accumulation of the newly identifiedmetabolites. Four of the nine inhibitors listed in Table 3 were profiled(FIG. 22 and FIG. 23). Except isovalerate, the levels of the other threeinhibitors were lower in the Low 4 AA condition compared to HIPDOGcondition. Leucine not being one of the amino acids which is controlledat low levels in the Low 4 AA condition, its metabolic intermediateisovalerate accumulates in Low 4 AA condition to levels similar to thoseseen the control HIPDOG condition. The cells in Low 4 AA conditions grewbetter peaking at cell densities 37×10⁶ cells/mL on day 9 of the culturewhereas the cell densities in control HIPDOG condition peaked around32×10⁶ cells/mL (FIG. 21A). Further, the increased growth observed inLow 4 AA condition translated into higher titer levels compared to thecontrol HIPDOG condition (FIG. 21B). Such an increase in the cell growthand productivity observed in the low amino acid condition can beexplained as an outcome of the reduced inhibitor biosynthesis andaccumulation (FIGS. 22 and 23).

Example 7: Use of RAMAN Spectroscopy for Online Measurement of the Four(Phenylalanine, Tyrosine, Tryptophan and Methionine) or Eight AminoAcids (Phenylalanine, Tyrosine, Tryptophan, Methionine, Leucine, Serine,Glycine, and Threonine) and Newly Identified Inhibitors

Raman spectroscopy is based on the inelastic scattering of monochromaticlight (photons) by a molecule. The technology uses the frequency shiftin the light, due to a change in energy of the photon when it isabsorbed and reemitted by the molecule, to determine the characteristicsof the molecule. This technology has been successfully used in microbialand mammalian cell culture bioreactors with success to measure thelevels of various process parameters. Raman spectroscopy has also beenused to determine the concentrations of glucose, lactate, ammonia,glutamine and glutamate in CHO cell cultures.

The Raman spectra for all the amino acids have been previously reportedin the literature. Raman spectra for the newly identified inhibitorymetabolites is characterized using a solution of the purifiedmetabolites. An empirical model is built by employing a training set ofspectral data generated using known concentrations of each of the fouror eight amino acids or the newly identified inhibitors, individually.The sample matrix (background) used for of the preparation ofcalibration samples (used as the training set) is a mix of spent mediasamples taken from different time points of different cell cultureprocesses. The model developed using such a training set is more generaland can be applied to any other cell culture process. This model is usedto measure the concentration of the four or eight amino acids or themetabolites (inhibitors) in the culture (online) and accordingly controlthe levels of amino acids through feedback control feeding strategies.

Example 8: Suppression of Inhibitor Formation Through Control of AminoAcids at Low Levels in Fed-Batch Cultures by Using Online Measurement ofInhibitor Concentration

A fed-batch process is designed to reduce the formation of theinhibitors through control of four (phenylalanine, tyrosine, tryptophanand methionine) or eight amino acids (phenylalanine, tyrosine,tryptophan, methionine, leucine, serine, glycine, and threonine) at lowlevels, for example between 0.2-1 mM in the cell culture medium. Such acontrol of the inhibitor production is attained by a feeding strategythat operates as a feed-back loop based on the online measurement of theinhibitors themselves. Such online measurements are in the form of RAMANspectroscopy or through use of HPLC/UPLC based technology with anauto-sampler that draws sample from reactor and transfers it to theequipment in a programmed manner. As and when the concentration of theinhibitors rises above a specified level (example: 0.2 mM), the amountof phenylalanine, tyrosine, tryptophan, methionine, leucine, serine,glycine, and threonine fed to the cells is decreased until theconcentration of inhibitors falls below a predefined level (for example0.1 mM).

Example 9: Suppression of Inhibitor Formation Through Online or OfflineMeasurement and Control of Amino Acids at Low Levels in Fed-BatchCultures

A fed-batch process is designed to reduce the formation of theinhibitors through control of the four (phenylalanine, tyrosine,tryptophan and methionine) or eight amino acids (phenylalanine,tyrosine, tryptophan, methionine, leucine, serine, glycine, andthreonine) at low levels (0.2-1 mM). Such a control of inhibitorformation is attained by a feeding strategy that operates as a feed-backloop based on the online measurement of the amino acids. Such onlinemeasurements is in the form of RAMAN spectroscopy or through use ofHPLC/UPLC based technology with an auto-sampler that draws sample fromreactor and transfers it to the equipment in a programmed manner. As andwhen the concentration of the amino acids falls below a specified level(example: 0.5 mM), estimated amounts of feed medium (similar to MediumB) is added to the culture so as to maintain the concentrations of thefour or eight amino acids.

Alternatively, the samples are taken on a once per day basis and aminoacid concentrations are measured offline using a UPLC/HPLC method. Theamino acid concentrations obtained are used to calculate the cellspecific uptake rates of the four (phenylalanine, tyrosine, tryptophanand methionine) or eight amino acids (phenylalanine, tyrosine,tryptophan, methionine, leucine, serine, glycine, and threonine) betweenprevious two sampling time points. Assuming that the cells maintain thesame specific rate of amino acid consumption until the next samplingtime point, the amount of feed medium (example: Medium B) to be addedtill the next sampling point is determined and provided to the cells,such that the concentrations of the amino acids are always within thedesired range (0.2 mM-1 mM).

Example 10: Suppression of Inhibitor Formation Through ProgrammedFeeding so as to Keep the Amino Acids at Low Levels in Fed-BatchCultures

The formation of the newly identified inhibitors in culture is kept lowby maintaining the concentration of the four (phenylalanine, tyrosine,tryptophan and methionine) or eight amino acids (phenylalanine,tyrosine, tryptophan, methionine, leucine, serine, glycine, andthreonine) at low levels in culture (0.2-1 mM). This is attained bydesigning a programmed feeding strategy (using modified Medium B) suchthat the concentrations of the amino acids, at any given time point inthe culture are always within the desired range (0.2 mM-1 mM). Such afeeding strategy is contrived by assessing the specific consumptionrates of amino acids at different time points along the culture andmodifying the concentration in the feed medium (Medium B) such that withthe above defined feeding strategy/schedule, sufficient amounts of aminoacids are provided to the culture to maintain the amino acidsconcentrations within the desired range (0.2-1 mM).

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EMBODIMENTS

1. A method of cell culture comprising(i) providing cells in a cell culture medium to start a cell cultureprocess, and,(ii) maintaining at least one metabolite selected from3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate,indolelactate, indolecarboxylic acid, homocysteine, 2-hydroxybutyricacid, isovalerate and formate below a concentration C1 in the cellculture medium, wherein C1 is 3 mM.2. The method of embodiment 1 wherein said metabolite is selected from3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate,indolelactate, indolecarboxylic acid, homocysteine and 2-hydroxybutyricacid3. The method of embodiment 1 wherein C1 is 1 mM.4. The method of embodiment 1 wherein C1 is 0.5 mM.5. The method of any one of embodiments 1 to 4 wherein step ii)comprises the step of measuring the concentration of said at least onemetabolite.6. The method of embodiment 5 wherein said concentration is measured offline.7. The method of embodiment 6 wherein measuring the concentration ofsaid at least one metabolite comprises taking a sample from the cellculture medium and measuring the concentration of said at least onemetabolite in said sample.8. The method of any one of embodiments 5 to 7 wherein saidconcentration is measured by LC-MS.9. The method of embodiment 5 wherein said concentration is measuredonline.10. The method of embodiment 9 wherein said concentration is measuredonline using Raman spectroscopy.11. The method of embodiment 9 wherein said concentration is measuredonline using NMR, HPLC or UPLC.12. The method of any one of embodiments 5 to 11 wherein saidconcentration is measured intermittently, every 30 min, every hour,every two hours, twice a day, daily, or every two days.13. The method of embodiment 9 or 10 wherein said concentration ismeasured continuously.14. The method of any one of embodiments 5 to 13 wherein, when themeasured concentration is above a predefined value, the concentration ofprecursor of said at least one metabolite in the cell culture medium isdecreased.15. The method of embodiment 14 wherein said predefined value is C1.16. The method of embodiment 14 wherein said predefined value is apercentage of C1.17. The method of embodiment 16 wherein said predefined value is 50, 55,60, 65, 70, 75, 80, 85, 90 or 95% of C1.18. The method of embodiment 17 wherein said predefined value is 80% ofC1.19. The method of any one of embodiments 14 to 18 wherein theconcentration of precursor is decreased by reducing the amount ofprecursor provided to the cells.20. The method of any one of embodiments 14 to 19 wherein theconcentration of precursor is decreased by reducing the concentration ofsaid precursor in the feed medium.21. The method of any one of embodiments 14 to 20 wherein theconcentration of precursor is decreased by reducing the feed rate.22. The method of any one of embodiments 14 to 21 wherein theconcentration of precursor is decreased by reducing the number of feeds.23. The method of any one of embodiments 14 to 22 wherein, when theconcentration of precursor is decreased by reducing the volume of feeds.24. The method of any one of embodiments 14 to 23 wherein when themeasured concentration of 3-(4-hydroxyphenyl)lactate,4-hydroxyphenylpyruvate and/or phenyllactate is above said predefinedvalue, the concentration of phenylalanine is decreased in the cellculture medium.25. The method of any one of embodiments 14 to 24 wherein when themeasured concentration of 3-(4-hydroxyphenyl)lactate and/or4-hydroxyphenylpyruvate is above said predefined value, theconcentration of tyrosine is decreased in the cell culture medium.26. The method of any one of embodiments 14 to 25 wherein when themeasured concentration of indolelactate and/or indolecarboxylic acid isabove said predefined value, the concentration of tryptophan isdecreased in the cell culture medium.27. The method of any one of embodiments 14 to 26 wherein when themeasured concentration of homocysteine and/or 2-hydroxybutyric acid isabove said predefined value, the concentration of methionine isdecreased in the cell culture medium.28. The method of any one of embodiments 14 to 27 wherein when themeasured concentration of isovalerate is above said predefined value,the concentration of leucine is decreased in the cell culture medium.29. The method of any one of embodiments 14 to 28 wherein when themeasured concentration of formate is above said predefined value, theconcentration of serine, threonine and/or glycine is decreased in thecell culture medium.30. The method of any one of embodiments 1 to 29 wherein step (ii)comprises maintaining 1, 2, 3, 4, 5, 6, 7, 8 or 9 of3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate,indolelactate, indolecarboxylic acid, homocysteine, 2-hydroxybutyricacid, isovalerate and formate below C1 in the cell culture medium.31. The method of any one of embodiments 1 to 30 wherein step (ii)comprises maintaining 1, 2, 3, 4, 5, 6, or 7 of3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate,indolelactate, indolecarboxylic acid, homocysteine and 2-hydroxybutyricacid below C1 in the cell culture medium.32. The method of any one of embodiments 1 to 31 wherein step (ii)comprises maintaining 3-(4-hydroxyphenyl)lactate,4-hydroxyphenylpyruvate and phenyllactate below C1 in the cell culturemedium.33. The method of any one of embodiments 1 to 32 wherein step (ii)comprises maintaining indolelactate and indolecarboxylic acid below C1in the cell culture medium.34. The method of any one of embodiments 1 to 33 wherein step (ii)comprises maintaining homocysteine and 2-hydroxybutyric acid below C1 inthe cell culture medium.35. The method of any one of embodiments 1 to 34 wherein step (ii)comprises maintaining isovalerate below C1 in the cell culture medium.36. The method of any one of embodiments 1 to 35 wherein step (ii)comprises maintaining formate below C1 in the cell culture medium.37. The method of any one of embodiments 1 to 36 wherein, in step (ii),the concentration of 3-(4-hydroxyphenyl)lactate is maintained below 0.5mM.38. The method of any one of embodiments 1 to 37 wherein, in step (ii),the concentration of 3-(4-hydroxyphenyl)lactate is maintained below 0.3mM.39. The method of any one of embodiments 1 to 38 wherein, in step (ii),the concentration of 3-(4-hydroxyphenyl)lactate is maintained below 0.1mM.40. The method of any one of embodiments 1 to 39 wherein, in step (ii),the concentration of 4-hydroxyphenylpyruvate is maintained below 0.1 mM.41. The method of any one of embodiments 1 to 40 wherein, in step (ii),the concentration of 4-hydroxyphenylpyruvate is maintained below 0.05mM.42. The method of any one of embodiments 1 to 41 wherein, in step (ii),the concentration of 4-hydroxyphenylpyruvate is maintained below 0.02mM.43. The method of any one of embodiments 1 to 42 wherein, in step (ii),the concentration of phenyllactate is maintained below 0.5 mM.44. The method of any one of embodiments 1 to 43 wherein, in step (ii),the concentration of phenyllactate is maintained below 0.2 mM.45. The method of any one of embodiments 1 to 44 wherein, in step (ii),the concentration of phenyllactate is maintained below 0.1 mM.46. The method of any one of embodiments 1 to 45 wherein, in step (ii),the concentration of indolelactate is maintained below 3 mM.47. The method of any one of embodiments 1 to 46 wherein, in step (ii),the concentration of indolelactate is maintained below 1 mM.48. The method of any one of embodiments 1 to 47 wherein, in step (ii),the concentration of indolelactate is maintained below 0.3 mM.49. The method of any one of embodiments 1 to 48 wherein, in step (ii),the concentration of indolelactate is maintained below 0.1 mM.50. The method of any one of embodiments 1 to 49 wherein, in step (ii),the concentration of indolecarboxylic acid is maintained below 1 mM.51. The method of any one of embodiments 1 to 50 wherein, in step (ii),the concentration of indolecarboxylic acid is maintained below 0.5 mM.52. The method of any one of embodiments 1 to 51 wherein, in step (ii),the concentration of indolecarboxylic acid is maintained below 0.2 mM.53. The method of any one of embodiments 1 to 52 wherein, in step (ii),the concentration of homocysteine is maintained below 0.5 mM.54. The method of any one of embodiments 1 to 53 wherein, in step (ii),the concentration of homocysteine is maintained below 0.3 mM.55. The method of any one of embodiments 1 to 54 wherein, in step (ii),the concentration of homocysteine is maintained below 0.1 mM.56. The method of any one of embodiments 1 to 55 wherein, in step (ii),the concentration of 2-hydroxybutyric acid is maintained below 1 mM.57. The method of any one of embodiments 1 to 56 wherein, in step (ii),the concentration of 2-hydroxybutyric acid is maintained below 0.5 mM.58. The method of any one of embodiments 1 to 57 wherein, in step (ii),the concentration of 2-hydroxybutyric acid is maintained below 0.2 mM.59. The method of any one of embodiments 1 to 58 wherein, in step (ii),the concentration of isovalerate is maintained below 2 mM.60. The method of any one of embodiments 1 to 59 wherein, in step (ii),the concentration of isovalerate is maintained below 1 mM.61. The method of any one of embodiments 1 to 60 wherein, in step (ii),the concentration of isovalerate is maintained below 0.5 mM.62. The method of any one of embodiments 1 to 61 wherein, in step (ii),the concentration of formate acid is maintained below 4 mM.63. The method of any one of embodiments 1 to 62 wherein, in step (ii),the concentration of formate acid is maintained below 3 mM.64. The method of any one of embodiments 1 to 63 wherein, in step (ii),the concentration of formate acid is maintained below 2 mM.65. A method of cell culture comprising(i) providing cells in a cell culture medium to start a cell cultureprocess, and,(ii) maintaining at least one amino acid selected from phenylalanine,tyrosine, tryptophan, methionine, leucine, serine, threonine and glycinebelow a concentration C2 in the cell culture medium, wherein C2 is 2 mM.66. The method of embodiment 65 wherein step ii) comprises the step ofmeasuring the concentration of said at least one amino acid.67. The method of embodiment 66 wherein said concentration is measuredoff line.68. The method of embodiment 67 wherein measuring the concentration ofsaid at least one amino acid comprises taking a sample from the cellculture medium and measuring the concentration of said at least oneamino acid in said sample.69. The method of any one of embodiments 66 to 68 wherein saidconcentration is measured by LC-MS.70. The method of embodiment 66 wherein said concentration is measuredonline.71. The method of embodiment 70 wherein said concentration is measuredonline using Raman spectroscopy.72. The method of embodiment 70 wherein said concentration is measuredonline using NMR, HPLC or UPLC.73. The method of any one of embodiments 66 to 72 wherein saidconcentration is measured intermittently, every 30 min, every hour,every two hours, twice a day, daily, or every two days.74. The method of embodiment 70 or 71 wherein said concentration ismeasured continuously.75. The method of any one of embodiments 65 to 74 wherein, when themeasured concentration is above a predefined value, the concentration ofsaid at least one amino acid in the cell culture medium is decreased.76. The method of embodiment 75 wherein said predefined value is C2.77. The method of embodiment 75 wherein said predefined value is apercentage of C2.78. The method of embodiment 77 wherein said predefined value is 50, 55,60, 65, 70, 75, 80, 85, 90 or 95% of C2.79. The method of embodiment 78 wherein said predefined value is 80% ofC2.80. The method of any one of embodiments 75 to 79 wherein, when theconcentration of said at least one amino acid is decreased by reducingthe amount of amino acid provided to the cells.81. The method of any one of embodiments 75 to 80 wherein, when theconcentration of said at least one amino acid is decreased by reducingthe concentration of said amino acid in the feed medium.82. The method of any one of embodiments 75 to 81 wherein, when theconcentration of said at least one amino acid is decreased by reducingthe feed rate.83. The method of any one of embodiments 75 to 82 wherein, when theconcentration of said at least one amino acid is decreased by reducingthe number of feeds.84. The method of any one of embodiments 75 to 83 wherein, when theconcentration of said at least one amino acid is decreased by reducingthe volume of feeds.85. The method of any one of embodiments 1 to 84 wherein theconcentration of phenylalanine is maintained below 2 mM in the cellculture medium.86. The method of any one of embodiments 1 to 85 wherein theconcentration of phenylalanine is maintained between 0.1 and 2 mM in thecell culture medium.87. The method of any one of embodiments 1 to 86 wherein theconcentration of phenylalanine is maintained between 0.1 and 1 mM in thecell culture medium.88. The method of any one of embodiments 1 to 87 wherein theconcentration of phenylalanine is maintained between 0.2 and 1 mM in thecell culture medium.89. The method of any one of embodiments 1 to 88 wherein theconcentration of phenylalanine is maintained between 0.5 and 1 mM in thecell culture medium.90. The method of any one of embodiments 1 to 89 wherein theconcentration of tyrosine is maintained below 2 mM in the cell culturemedium.91. The method of any one of embodiments 1 to 90 wherein theconcentration of tyrosine is maintained between 0.1 and 2 mM in the cellculture medium.92. The method of any one of embodiments 1 to 91 wherein theconcentration of tyrosine is maintained between 0.1 and 1 mM in the cellculture medium.93. The method of any one of embodiments 1 to 92 wherein theconcentration of tyrosine is maintained between 0.2 and 1 mM in the cellculture medium.94. The method of any one of embodiments 1 to 93 wherein theconcentration of tyrosine is maintained between 0.5 and 1 mM in the cellculture medium.95. The method of any one of embodiments 1 to 94 wherein theconcentration of tryptophan is maintained below 2 mM in the cell culturemedium.96. The method of any one of embodiments 1 to 95 wherein theconcentration of tryptophan is maintained between 0.1 and 2 mM in thecell culture medium.97. The method of any one of embodiments 1 to 96 wherein theconcentration of tryptophan is maintained between 0.1 and 1 mM in thecell culture medium.98. The method of any one of embodiments 1 to 97 wherein theconcentration of tryptophan is maintained between 0.2 and 1 mM in thecell culture medium.99. The method of any one of embodiments 1 to 98 wherein theconcentration of tryptophan is maintained between 0.5 and 1 mM in thecell culture medium.100. The method of any one of embodiments 1 to 99 wherein theconcentration of methionine is maintained below 2 mM in the cell culturemedium.101. The method of any one of embodiments 1 to 100 wherein theconcentration of methionine is maintained between 0.1 and 2 mM in thecell culture medium.102. The method of any one of embodiments 1 to 101 wherein theconcentration of methionine is maintained between 0.1 and 1 mM in thecell culture medium.103. The method of any one of embodiments 1 to 102 wherein theconcentration of methionine is maintained between 0.2 and 1 mM in thecell culture medium.104. The method of any one of embodiments 1 to 103 wherein theconcentration of methionine is maintained between 0.5 and 1 mM in thecell culture medium.105. The method of any one of embodiments 1 to 104 wherein theconcentration of leucine is maintained below 2 mM in the cell culturemedium.106. The method of any one of embodiments 1 to 105 wherein theconcentration of leucine is maintained between 0.1 and 2 mM in the cellculture medium.107. The method of any one of embodiments 1 to 106 wherein theconcentration of leucine is maintained between 0.1 and 1 mM in the cellculture medium.108. The method of any one of embodiments 1 to 107 wherein theconcentration of leucine is maintained between 0.2 and 1 mM in the cellculture medium.109. The method of any one of embodiments 1 to 108 wherein theconcentration of leucine is maintained between 0.5 and 1 mM in the cellculture medium.110. The method of any one of embodiments 1 to 109 wherein theconcentration of serine is maintained below 2 mM in the cell culturemedium.111. The method of any one of embodiments 1 to 110 wherein theconcentration of serine is maintained between 0.1 and 2 mM in the cellculture medium.112. The method of any one of embodiments 1 to 111 wherein theconcentration of serine is maintained between 0.1 and 1 mM in the cellculture medium.113. The method of any one of embodiments 1 to 112 wherein theconcentration of serine is maintained between 0.2 and 1 mM in the cellculture medium.114. The method of any one of embodiments 1 to 113 wherein theconcentration of serine is maintained between 0.5 and 1 mM in the cellculture medium.115. The method of any one of embodiments 1 to 114 wherein theconcentration of threonine is maintained below 2 mM in the cell culturemedium.116. The method of any one of embodiments 1 to 115 wherein theconcentration of threonine is maintained between 0.1 and 2 mM in thecell culture medium.117. The method of any one of embodiments 1 to 116 wherein theconcentration of threonine is maintained between 0.1 and 1 mM in thecell culture medium.118. The method of any one of embodiments 1 to 117 wherein theconcentration of threonine is maintained between 0.2 and 1 mM in thecell culture medium.119. The method of any one of embodiments 1 to 118 wherein theconcentration of threonine is maintained between 0.5 and 1 mM in thecell culture medium.120. The method of any one of embodiments 1 to 119 wherein theconcentration of glycine is maintained below 2 mM in the cell culturemedium.121. The method of any one of embodiments 1 to 120 wherein theconcentration of glycine is maintained between 0.1 and 2 mM in the cellculture medium.122. The method of any one of embodiments 1 to 121 wherein theconcentration of glycine is maintained between 0.1 and 1 mM in the cellculture medium.123. The method of any one of embodiments 1 to 122 wherein theconcentration of glycine is maintained between 0.2 and 1 mM in the cellculture medium.124. The method of any one of embodiments 1 to 123 wherein theconcentration of glycine is maintained between 0.5 and 1 mM in the cellculture medium.125. The method of any one of embodiments 1 to 124 wherein the cellculture medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 ofglycine, valine, leucine, isoleucine, proline, serine, threonine,lysine, arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 1 mM, 1.5 mM, 2 mM, 3 mM or 5 mM.126. The method of any one of embodiments 1 to 124 wherein the cellculture medium comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 of valine,isoleucine, proline, lysine, arginine, histidine, aspartate, glutamateand asparagine at a concentration above 1 mM, 1.5 mM, 2 mM, 3 mM or 5mM.127. The method of any one of embodiments 1 to 124 wherein the cellculture medium comprises one of glycine, valine, leucine, isoleucine,proline, serine, threonine, lysine, arginine, histidine, aspartate,glutamate and asparagine at a concentration above 2 mM.128. The method of any one of embodiments 1 to 124 wherein the cellculture medium comprises one of valine, isoleucine, proline, lysine,arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 2 mM.129. The method of any one of embodiments 1 to 124 wherein the cellculture medium comprises one of glycine, valine, leucine, isoleucine,proline, serine, threonine, lysine, arginine, histidine, aspartate,glutamate and asparagine at a concentration above 5 mM.130. The method of any one of embodiments 1 to 124 wherein the cellculture medium comprises one of valine, isoleucine, proline, lysine,arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 5 mM.131. The method of any one of embodiments 1 to 130 where lactate ismaintained at low levels in the cell culture medium.132. The method of any one of embodiments 1 to 131 where concentrationof lactate in the cell culture medium is maintained below 90 mM.133. The method of any one of embodiments 1 to 132 where concentrationof lactate in the cell culture medium is maintained below 70 mM.134. The method of any one of embodiments 1 to 133 where concentrationof lactate in the cell culture medium is maintained below 50 mM.135. The method of any one of embodiments 1 to 134 where concentrationof lactate in the cell culture medium is maintained below 40 mM.136. The method of any one of embodiments 1 to 135 where concentrationof lactate in the cell culture medium is maintained at low levels bycontrolling the amount of glucose provided to the cell culture.137. The method of any one of embodiments 1 to 136 where concentrationof lactate in the cell culture medium is maintained at low levels byusing the HIPDOG process.138. The method of any one of embodiments 1 to 137 wherein a pH sensoris used to monitor pH of the cell culture, and, in response to a riseabove a predetermined pH value, glucose is fed to the cell culture.139. The method of embodiment 138, wherein the predetermined pH value isapproximately 7.140. The method of any one of embodiments 1 to 139 where ammonia iscontrolled at low levels in the cell culture medium.141. The method of any one of embodiments 1 to 140 where concentrationof ammonia in the cell culture medium is maintained below 20 mM.142. The method of any one of embodiments 1 to 141 where concentrationof ammonia in the cell culture medium is maintained below 10 mM.143. The method of any one of embodiments 1 to 142 where concentrationof ammonia in the cell culture medium is maintained below 8 mM.144. The method of any one of embodiments 1 to 143, wherein the cellsare mammalian cells.145. The method embodiment 144, wherein the mammalian cells are selectedfrom BALB/c mouse myeloma line, human retinoblasts (PER.C6), monkeykidney cells, human embryonic kidney line (293), baby hamster kidneycells (BHK), Chinese hamster ovary cells (CHO), mouse sertoli cells,African green monkey kidney cells (VERO-76), human cervical carcinomacells (HeLa), canine kidney cells, buffalo rat liver cells, human lungcells, human liver cells, mouse mammary tumor cells, TRI cells, MRC 5cells, FS4 cells, or human hepatoma line (Hep G2).146. The method of embodiment 145, wherein the mammalian cells are CHOcells.147. The method of embodiment 146, wherein the mammalian cells areGS-CHO cells.148. The method of any one of embodiments 1 to 147, wherein the cellculture is a fed batch culture.149. The method of embodiment 148, wherein the fed batch culturecomprises a base medium supplemented with feed media.150. The method of embodiment 148 or 149, wherein the base medium and/orfeed media are substantially free of serum.151. The method of any one of embodiments 1 to 150 wherein said methodis a method for improving cell growth.152. The method of any one of embodiments 1 to 151 wherein said methodis a method for improving cell growth in high density cell culture.153. The method of any one of embodiments 1 to 152 wherein said methodis a method for improving cell growth in a cell culture where maximumviable cell density is above 5×10⁶ cells/mL, and preferably above 5×10⁷cells/mL.154. The method of any one of embodiments 1 to 153, wherein the cellculture system is a large-scale production system.155. The method of any one of embodiments 1 to 154, wherein the cellculture system uses a bioreactor.156. The method of any one of embodiments 1 to 155, wherein the cellculture method comprises a growth phase and a production phase and step(ii) is applied during the growth phase.157. The method of any one of embodiments 1 to 156, wherein the volumeof the cell culture medium is at least 500 L.158. The method of any one of embodiments 1 to 156, wherein the volumeof the cell culture medium is at least 5000 L.159. The method of any one of embodiments 1 to 158, wherein the cellsexpress a recombinant protein.160. The method of embodiment 159, wherein the recombinant protein isselected from the group consisting of antibodies or fragments thereof,nanobodies, single domain antibodies, Small ModularImmunoPharmaceuticals™ (SMIPs), VHH antibodies, camelid antibodies,shark single domain polypeptides (IgNAR), single domain scaffolds (e.g.,fibronectin scaffolds), SCORPION™ therapeutics (single chainpolypeptides comprising an N-terminal binding domain, an effectordomain, and a C-terminal binding domain), growth factors, clottingfactors, cytokines, fusion proteins, pharmaceutical drug substances,vaccines, enzymes and combinations thereof.161. The method of embodiment 159 or 160, wherein the recombinantprotein is a glycoprotein.162. The method of any one of embodiments 159 to 161, further comprisingobtaining recombinant protein produced by the cells.163. The method of embodiment 162, further comprising purifying therecombinant protein.164. The method of embodiment 163, further comprising preparing apharmaceutical composition comprising the recombinant protein.165. A recombinant protein produced using a method of any one ofembodiments 159 to 163.166. The method of any one of embodiments 1 to 130, wherein cell growthand/or productivity are increased as compared to a control culture, saidcontrol culture being identical except it does not comprise step (ii).167. The method of embodiment 166, wherein the cell growth is determinedby viable cell density, maximum viable cell density or Integrated ViableCell Count.168. The method of embodiment 166 or 167, wherein the cell growth isdetermined by maximum cell density.169. The method of any one of embodiments 166 to 168, wherein the cellgrowth is increased by at least 5% as compared to the control culture.170. The method of any one of embodiments 166 to 169, wherein the cellgrowth is increased by at least 10% as compared to the control culture.171. The method of any one of embodiments 166 to 170, wherein the cellgrowth is increased by at least 20% as compared to the control culture.172. The method of embodiment 166, wherein the productivity isdetermined by titer, and/or volumetric productivity.173. The method of any one of embodiments 166-172, wherein theproductivity is determined by titer.174. The method of embodiments 172 or 173, wherein the productivity isincreased by at least 5% as compared to the control culture.175. The method of any one of embodiments 172 to 174, wherein theproductivity is increased by at least 10% as compared to the controlculture.176. The method of any one of embodiments 172 to 175, wherein theproductivity is increased by at least 20% as compared to the controlculture.177. The method of any one of embodiments 1 to 164 and 166 to 176,wherein the maximum viable cell density of the cell culture is greaterthan 1×10⁶ cells/mL, 5×10⁶ cells/mL, 1×10⁷ cells/mL, 5×10⁷ cells/mL,1×10⁸ cells/mL or 5×10⁸ cells/mL.178. The method of embodiment 177, wherein the maximum viable celldensity of the cell culture is greater than 5×10⁶ cells/mL andpreferably greater than 5×10⁷ cells/mL.179. The method of embodiment 178, wherein the maximum viable celldensity of the cell culture is greater than 1×10⁸ cells/mL cells/mL.180. A cell culture medium comprising phenylalanine at a concentrationbelow 2 mM.181. The cell culture medium of embodiment 180 comprising tyrosine at aconcentration below 2 mM.182. The cell culture medium of embodiments 180 or 181 comprisingtryptophan at a concentration below 2 mM.183. A cell culture medium of any one of embodiments 180 to 182comprising methionine at a concentration below 2 mM.184. The cell culture medium of any one of embodiments 180 to 183comprising leucine at a concentration below 2 mM.185. The cell culture medium of any one of embodiments 180 to 184comprising serine at a concentration below 2 mM.186. The cell culture medium of any one of embodiments 180 to 185comprising threonine at a concentration below 2 mM.187. The cell culture medium of any one of embodiments 180 to 186comprising glycine at a concentration below 2 mM.188. A cell culture medium comprising tyrosine at a concentration below2 mM.189. The cell culture medium of embodiment 188 comprising tryptophan ata concentration below 2 mM.190. The cell culture medium of embodiments 188 or 189 comprisingmethionine at a concentration below 2 mM.191. The cell culture medium of any one of embodiments 188 to 190comprising leucine at a concentration below 2 mM.192. The cell culture medium of any one of embodiments 188 to 190comprising serine at a concentration below 2 mM.193. The cell culture medium of any one of embodiments 188 to 190comprising threonine at a concentration below 2 mM.194. The cell culture medium of any one of embodiments 188 to 190comprising glycine at a concentration below 2 mM.195. A cell culture medium comprising tryptophan at a concentrationbelow 2 mM.196. The cell culture medium of embodiment 195 comprising methionine ata concentration below 2 mM.197. The cell culture medium of any one of embodiments 195 to 196comprising leucine at a concentration below 2 mM.198. The cell culture medium of any one of embodiments 195 to 197comprising serine at a concentration below 2 mM.199. The cell culture medium of any one of embodiments 195 to 198comprising threonine at a concentration below 2 mM.200. The cell culture medium of any one of embodiments 195 to 199comprising glycine at a concentration below 2 mM.201. A cell culture medium comprising methionine at a concentrationbelow 2 mM.202. The cell culture medium of embodiment 201 comprising leucine at aconcentration below 2 mM.203. The cell culture medium of any one of embodiments 201 to 202comprising serine at a concentration below 2 mM.204. The cell culture medium of any one of embodiments 201 to 203comprising threonine at a concentration below 2 mM.205. The cell culture medium of any one of embodiments 201 to 204comprising glycine at a concentration below 2 mM.206. A cell culture medium comprising leucine at a concentration below 2mM.207. The cell culture medium of embodiment 206 comprising serine at aconcentration below 2 mM.208. The cell culture medium of any one of embodiments 206 to 207comprising threonine at a concentration below 2 mM.209. The cell culture medium of any one of embodiments 206 to 208comprising glycine at a concentration below 2 mM.210. A cell culture medium comprising serine at a concentration below 2mM.211. The cell culture medium of embodiment 210 comprising threonine at aconcentration below 2 mM.212. The cell culture medium of any one of embodiments 210 to 211comprising glycine at a concentration below 2 mM.213. A cell culture medium comprising threonine at a concentration below2 mM.214. The cell culture medium of any one of embodiments 210 to 213comprising glycine at a concentration below 2 mM.215. A cell culture medium comprising glycine at a concentration below 2mM.216. The cell culture medium of any one of embodiments 180 to 215wherein said concentration is below 1 mM.217. The cell culture medium of any one of embodiments 180 to 215wherein said concentration is between 0.1 and 2 mM.218. The cell culture medium of any one of embodiments 180 to 215wherein said concentration is between 0.1 and 1 mM.219. The cell culture medium of any one of embodiments 180 to 215wherein said concentration is between 0.2 and 1 mM.220. The cell culture medium of any one of embodiment 180 to 215 whereinsaid concentration is between 0.5 and 1 mM.221. The cell culture medium of any one of embodiment 180 to 220 whereinsaid medium further comprises at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 ofvaline, isoleucine, proline, lysine, arginine, histidine, aspartate,glutamate and asparagine at a concentration above 2 mM.222. The cell culture medium of any one of embodiment 180 to 220 whereinsaid medium comprises at least 5 of valine, isoleucine, proline, lysine,arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 2 mM.223. The cell culture medium of any one of embodiment 180 to 220 whereinsaid medium comprises valine, isoleucine, proline, lysine, arginine,histidine, aspartate, glutamate and asparagine at a concentration above2 mM.224. The cell culture medium of any one of embodiment 180 to 220 whereinsaid medium comprises valine, isoleucine, proline, lysine, arginine,histidine, aspartate, glutamate and asparagine at a concentration above1 mM.225. The cell culture medium of any one of embodiment 221 to 224 whereinsaid concentration is above 3 mM.226. The cell culture medium of any one of embodiment 180 to 225 whereinsaid medium comprises valine at a concentration above 3 mM.227. The cell culture medium of any one of embodiment 180 to 226 whereinsaid medium comprises valine at a concentration above 5 mM.228. The cell culture medium of any one of embodiment 180 to 227 whereinsaid medium comprises valine at a concentration above 10 mM.229. The cell culture medium of any one of embodiment 180 to 228 whereinsaid medium comprises isoleucine at a concentration above 3 mM.230. The cell culture medium of any one of embodiment 180 to 229 whereinsaid medium comprises isoleucine at a concentration above 5 mM.231. The cell culture medium of any one of embodiment 180 to 230 whereinsaid medium comprises isoleucine at a concentration above 10 mM.232. The cell culture medium of any one of embodiment 180 to 231 whereinsaid medium comprises cysteine at a concentration above 1.5 mM, andpreferably above 3 mM.233. The cell culture medium of any one of embodiment 180 to 232 whereinsaid medium comprises cysteine at a concentration above 5 mM.234. The cell culture medium of any one of embodiment 180 to 233 whereinsaid medium comprises cysteine at a concentration above 10 mM.235. The cell culture medium of any of embodiments 180 to 234 for use ina method of any one of embodiments 1 to 164 and 166 to 179.236. The cell culture medium of any of embodiments 180 to 234 for use asa base medium in a method of any one of embodiments 1 to 164 and 166 to179.237. The cell culture medium of any of embodiments 180 to 234 for use asa feed medium in a method of any one of embodiments 1 to 164 and 166 to179.238. The method of any one of embodiments 1 to 164 and 166 to 179wherein the medium of embodiments 146 to 184 is used as a feed medium.239. The method of any one of embodiments 1 to 164 and 166 to 179wherein the medium of embodiments 146 to 184 is used as a base medium.

1. A method of cell culture comprising (i) providing cells in a cellculture medium to start a cell culture process, and, (ii) maintaining atleast one metabolite selected from 3-(4-hydroxyphenyl)lactate,4-hydroxyphenylpyruvate, phenyllactate, indolelactate, indolecarboxylicacid, homocysteine, 2-hydroxybutyric acid, isovalerate and formate belowa concentration C1 in the cell culture medium, wherein C1 is 3 mM. 2.The method of claim 1 wherein C1 is 1 mM.
 3. The method of claim 1wherein step ii) comprises the step of measuring the concentration ofsaid at least one metabolite, and, when the measured concentration isabove a predefined value, the concentration of precursor of said atleast one metabolite in the cell culture medium is decreased by reducingthe amount of precursor provided to the cells.
 4. The method of claim 3wherein said concentration of said at least one metabolite is measuredonline using NMR, HPLC or UPLC.
 5. The method of claim 3 wherein, whenthe measured concentration of 3-(4-hydroxyphenyl)lactate,4-hydroxyphenylpyruvate and/or phenyllactate is above said predefinedvalue, the concentration of phenylalanine is decreased in the cellculture medium and/or, when the measured concentration of3-(4-hydroxyphenyl)lactate and/or 4-hydroxyphenylpyruvate is above saidpredefined value, the concentration of tyrosine is decreased in the cellculture medium, and/or, when the measured concentration of indolelactateand/or indolecarboxylic acid is above said predefined value, theconcentration of tryptophan is decreased in the cell culture mediumand/or, when the measured concentration of homocysteine and/or2-hydroxybutyric acid is above said predefined value, the concentrationof methionine is decreased in the cell culture medium, and/or, when themeasured concentration of isovalerate is above said predefined value,the concentration of leucine is decreased in the cell culture medium,and/or, when the measured concentration of formate is above saidpredefined value, the concentration of serine, threonine and/or glycineis decreased in the cell culture medium.
 6. The method of claim 1wherein step (ii) comprises maintaining 2, 3, 4, 5, 6, 7, 8 or 9 of3-(4-hydroxyphenyl)lactate, 4-hydroxyphenylpyruvate, phenyllactate,indolelactate, indolecarboxylic acid, homocysteine, 2-hydroxybutyricacid, isovalerate and formate below C1 in the cell culture medium.
 7. Amethod of cell culture comprising (i) providing cells in a cell culturemedium to start a cell culture process, and, (ii) maintaining at leastone amino acid selected from phenylalanine, tyrosine, tryptophan,methionine, leucine, serine, threonine and glycine below a concentrationC2 in the cell culture medium, wherein C2 is 2 mM.
 8. The method ofclaim 7 wherein step ii) comprises the step of measuring theconcentration of said at least one amino acid and when the measuredconcentration is above a predefined value, the concentration of said atleast one amino acid in the cell culture medium is decreased by reducingthe amount of amino acid provided to the cells.
 9. The method of claim 8wherein said predefined value is C2 or 50, 55, 60, 65, 70, 75, 80, 85,90 or 95% of C2.
 10. The method of claim 7 wherein the concentration ofphenylalanine, tyrosine, tryptophan, methionine, leucine, serine,threonine, glycine is maintained below 2 mM in the cell culture medium.11. The method of claim 1 wherein the cell culture medium comprises 1,2, 3, 4, 5, 6, 7, 8 or 9 of valine, isoleucine, proline, lysine,arginine, histidine, aspartate, glutamate and asparagine at aconcentration above 1 mM, 1.5 mM, 2 mM, 3 mM or 5 mM.
 12. The method ofclaim 1 wherein a pH sensor is used to monitor pH of the cell culture,and, in response to a rise above a predetermined pH value, glucose isfed to the cell culture.
 13. The method of claim 1, wherein the cellsare CHO cells.
 14. The method of claim 1, wherein the cell culture is afed batch culture.
 15. The method of claim 1, wherein the cells expressa recombinant protein.
 16. The method of claim 15, further comprisingobtaining and purifying the recombinant protein produced by the cells.17. The method of claim 1, wherein cell growth and/or productivity areincreased as compared to a control culture, said control culture beingidentical except it does not comprise step (ii).
 18. The method of claim17, wherein the cell growth is determined by maximum viable cell densityand is increased by at least 5% as compared to the control culture. 19.The method of claim 17, wherein the productivity is determined by titerand is increased by at least 5% as compared to the control culture. 20.The method of claim 1, wherein the maximum viable cell density of thecell culture is greater than 1×10⁶ cells/mL, 5×10⁶ cells/mL, 1×10⁷cells/mL, 5×10⁷ cells/mL, 1×10⁸ cells/mL or 5×10⁸ cells/mL.
 21. Themethod of claim 1, wherein the cell culture method comprises a growthphase and a production phase and step (ii) is applied during the growthphase.
 22. A cell culture medium comprising 1, 2, 3, 4, 5, 6, 7 or 8 ofphenylalanine, tyrosine, tryptophan, methionine, leucine, serine,threonine or glycine at a concentration below 2 mM.
 23. The cell culturemedium of claim 22 wherein said concentration is between 0.5 and 1 mM.24. The cell culture medium of claim 22 wherein said medium furthercomprises at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 of valine, isoleucine,proline, lysine, arginine, histidine, aspartate, glutamate andasparagine at a concentration above 2 mM.
 25. The cell culture medium ofclaim 22 for use in a method of claim 1.